LIBRARY 

OF    THE 

UNIVERSITY  OF  CALIFORNIA. 


BIOLOGY 

LIBRARY 

G 


Ocular  or  Eye  piece 


Draw-Tube 


Rack  and  Pinion  for 
coarse  adjustment 


Micrometer 
Screw  for 

fine  adjustment 


Abbe  Condenser 
Iris  Diaphragm 


DIRECTIONS 


FOR 


LABORATORY  WORK 


IN 


BACTERIOLOGY 


FOR  THE  USE  OF  THE  MEDICAL  CLASSES 
IN  THE  UNIVERSITY  OF  MICHIGAN. 


FREDERICK  G.  NOVY,  Se.D.,  M.D., 

JUNIOR   PROFESSOR   OF   HYGIENE  AND   PHYSIOLOGICAL   CHEMISTRY. 


GEORGE  WAHR: 

PUBLISHER    AND    BOOKSELLER, 

ANN   ARBOR,    MICH. 


:  /  7 


Li!-'       - 


Copyrighted  1894. 

GEORGE   \VAHK. 


ANN  ARBOR  COURIER, 

PRINTERS  AND  BINDERS. 


PREFACE. 


No  attempt  has  been  made  in  the  following  pages  at  a  formal, 
systematic  presentation  of  Bacteriology.  The  subject-matter  has 
been  arranged  entirely  with  reference  to  progressive  work  in  the 
laboratory  and,  more  especially,  corresponds  with  the  work  as 
carried  on  in  the  Hygienic  Laboratory  of  the  University  of  Michigan. 
The  course  covers  a  period  of  twelve  weeks  of  daily  afternoon  work. 
Illustrations  of  the  various  bacteria  and  of  their  cultural  character- 
istics have  been  expressly  omitted,  as  the  student  is  expected  to 
sketch  from  observation  the  form  of  .each  organism  and  its  peculiar- 
ities of  growth  in  the  colony,  and  in  tube  culture.  Blank  pages  are 
provided  for  this  purpose  and  for  such  additional  notes  as  may  bo 
desirable. 

The  works  that  have  been  drawn  upon  freely  in  the  preparation 
of  these  pages  are  Fraeukel's  Grundriss  der  Bakterienkunde, 
Eisenberg's  Bakteriologische  Diagnostik,  and  Fliigge's  Die  Mikro- 
organisnien.  The  larger  works  of  Baumgarten  and  of  Sternberg  were 
likewise  frequently  consulted,  and  in  many  instances  recourse  was 
had  to  the  original  sources. 

For  the  frontispiece  plate,  which  is  intended  to  show  the  com- 
ponent parts  of  the  microscope,  I  am  indebted  to  the  firm  of  E. 
Leitz,  of  Wetzlar. 

F.  G.  NOVY. 
ANN  ARBOR,  April,  1894. 


132699 


LABORATORY  Y/ORK  IN  BACTERIOLOGY. 

i. 

FORM  AND  CLASSIFICATION. 

Bacteria  as  single-celled  microscopic  plants. 

.Relation  to  algae — to  fungi — to  yeasts. 
Classified  according  to  external  form  into — 
Micrococcus — spherical. 
Bacillus — rod-shaped. 
Spirillum — screw-shaped. 

The  term  bacterium  still  used  occasionally  to  desig- 
nate a  very  short  bacillus. 

Vibrio — is  the  term  applied  to  organisms  which 
may  form  spirals,  but  commonly  grow  in  seg- 
ments of  a  spiral  giving  rise  to  comma — or 
^— N  shaped  forms. 

Lack  of  a  natural  classification  of  bacteria. 
Variation  in  form  as  a  result  of  natural  conditions — 
environment. 

Young  and  fully  developed  cells. 

Nature  of  the  medium  on  whhh  the  growth  occurs. 

Solid  and  liquid  media. 

Temperature. 

Unfavorable  media  give  rise  to  involution  forms 

— degenerations. 

Variations     resulting    from     artificial     conditions  — 
methods  of  examination. 

Deposition  of  aniline  dyes.     Simple  and  double 
stains,  as  of  tubercle  and  of  leprosy  bacillus. 
Contraction  of  protoplasm  by  alcohol — by  iodine. 
Action  of  heat. 

Constancy  of  form  and  of  species. 
Demonstration  of,  Micrococcup, 
Bacillus, 
Spirillum. 


PREPARATION  OF  NUTRIENT  GELATIN. 

LABORATORY  WORK. — Place  500  g.  of  chopped  lean  beef 
into  a  clean  1^-2  litre  flask;  add  1000  c.  c.  of  tap-water 
and  insert  a  cotton  plug  into  the  mouth  of  the  flask.  Shake 
the  flask  repeatedly  during  the  next  15-30  rain.,  and  then 
immerse  in  boiling  water  in  a  water-bath  and  heat  for 
15-30  min.  Filter  through  muslin  and  to  the  filtrate  or 
meat  extract  thus  obtained  add, 

100  g.  of  gelatin 
10  g.  of  dry  peptone 
5  g.  of  common  salt. 

Warm  in  the  water-bath  until  the  gelatin  melts,  then 
neutralize  or  render  slightly  alkaline  by  cautious  addi- 
tions of  a  saturated  sodium  carbonate  solution  (avoid 
excess  of  alkali).  Continue  heating  in  a  water-bath,  with 
occasional  shaking,  for  f  to  1  hr.  Filter  through  a  plaited 
filter.  The  filtrate  should  be,  (1)  perfectly  clear,  (2) 
should  be  neutral  or  slightly  alkaline  in  reaction,  (3) 
should  not  become  cloudy  or  coagulate  when  boiled  in  a 
test  tube  for  1  to  2  minutes,  (4)  should  solidify  when 
cooled. 

If  the  filtrate  is  cloudy  and  strongly  alkaline  correct 
the  reaction  by  the  addition  of  dilute  acetic  acid.  If  it 
becomes  cloudy  or  coagulates  when  warmed,  continue 
heating  in  the  water-bath. 

If  the  filtered  gelatin  answers  the  above  requirements 
then  fill,  with  the  aid  of  a  small  funnel,  into  test-tubes  to 
a  depth  of  1  to  1^  inches.  Avoid  touching  the  neck  of 
the  tube  with  gelatin.  These  test-tubes  are  first 
thoroughly  cleansed,  allowed  to  dry,  then  plugged  with 
cotton,  placed  upright  in  a  wire  basket  and  sterilized  in  a 
dry  heat  oven  at  150  to  175°  C.  for  1  hour. 

After  the  tubes  have  been  filled  with  gelatin  they  are 
again  sterilized  by  heating  in  the  steam  sterilizer,  (100° 
C.)  for  15  minutes,  each  day,  for  the  next  consecutive 
three  days. 


MEMORANDA. 


MEMORANDA. 


II. 

STRUCTURE  OF  THE  BACTERIAL  CELL. 

The  cell- wall  possibly  composed  of  cellulose  or  woody- 
fibre 

Difficultly  seen  —  its    demonstration    by   icdine. 

Capsulated  bacteria. 
ZoOgloea — the  result  of  fusion  of  the  cell-walls  of 

many  bacteria. 

The  cell  contents — protoplasm — existence  of  a  nucleus. 
Homogeneous — granular. 
As  a  rule  the  cell  is  colorless.     A  few  are  slightly 

colored. 
Chlorophyll,  the  green  coloring  matter  of  plants, 

is  present  in  a  siunll  number. 
The    granulose    reaction    given    by    some  — blue 
color  with  iodine,  same  as  with  boiled  starch. 
Motion — Brownian,  or    molecular   movement.     Real 
motion. 

Actual  motion  observed  in  many  bacilli,  in  spi- 
rilla and  in  two  micrococci. 
Whips   or  flagella.      Their  arrangement   on    the 

bacillus, spirillum,  vibrios,  micrococciis. 
Giant  whip-. 
Demonstration  of, 

Action  of  iodine  on  protoplasm, 

Capsules, 

Whips. 

PREPARATION  OF  POTATO  CULTURES. 

Select  three  sound  potatoes  and  then  clean  them 
thoroughly,  under  the  tap,  with  the  aid  of  a  brush.  By 
means  of  a  knife  remove  any  bad  spots  or  depressions  that 


—  8  — 

may  exist,  since  these  frequently  harbor  bacteria  which 
are  highly  resistant  to  destruction.  Place  the  potatoes, 
thus  prepared,  in  a  solution  of  mercuric  chloride  (1-1000) 
for  |  to  1  hour;  transfer  to  a  tin  pail  with  a  perforated 
bottom,  and  then  place  tin's  in  a  steam  sterilizer.  Heat 
for  f  to  1  hour, — the  potatoes  should  be  well  cooked. 
Remove  the  pail  and  allow  the  potatoes  to  partially  cool. 

A  "moist  chamber"  is  prepared  by  placing  around 
filter  paper  on  the  bottom  of  the  lower  dish  and  moisten- 
ing it  with  mercuric  chloride,  allowing  any  excess  of  the 
solution  to  drain  off.  Three  potato  knives  are  sterilized 
by  heating  in  the  flame  till  the  edge  begins  to  redden. 
They  are  then  set  aside  to  cool,  with  the  edge  up,  on  a 
block  or  over  the  edge  of  the  table  so  that  the  blade  does 
not  touch  anything. 

The  partially  cooled  potato  is  now  picked  up  with  the 
left  hand,  which  previously  has  been  dipped  in  mercuric 
chloride,  and  cut  into  halves  by  a  horizontal  section  with 
a  sterilized  knife.  Each  half  of  the  potato  is  carefully 
placed  in  the  moist  chamber  with  the  cut  surface  turned 
upwards.  Contact  with  the  cut  surface  must  be  avoided. 

To  inoculate,  transfer  a  small  portion  of  the  bacterial 
growth  by  means  of  a  sterilized  and  cooled  wire  or  knife 
to  the  potato,  and  then  thoroughly  spread  this  material 
over  the  surface  avoiding  the  outer  £  inch.  The  potato 
is  held  in  the  fingers  of  the  left  hand,  which  has  been 
dipped  in  mercuric  chloride.  The  number  of  bacteria 
which  is  thus  transferred  to  the  surface  of  the  potato  is 
usually  so  great  that  when  they  develop  the  entire  surface 
is  covered  with  a  continuous  growth.  To  isolate  bac- 
teria, therefore,  it  is  necessary  to  resort  to  dilution  cul- 
ture. 

For  this  purpose  a  small  amount  of  material  is  taken 
from  the  surface  of  the  inoculated  potato,  or  No.  1,  by 
means  of  a  sterilized  knife,  and  transferred  in  the  same 
manner  as  before  to  the  surface  of  potato  No.  2,  where  it  is 
likewise  spread  thoroughly,  and  evenly,  over  the  surface. 
The  number  of  bacteria  transplanted  to  the  second  potato 


MEMORANDA. 


MEMORANDA. 


-9- 

is  still,  as  a  rule,  too  great.  Hence,  with  a  sterilized  knife 
a  minute  amount  of  material  is  taken  from  the  surface  of 
potato  No.  2  and  transferred  to  that  of  potato  No.  3,  where 
it  is  spread  as  before. 

If  the  inoculation  is  properly  carried  out  the  third 
potato  will  have-but  a  few  bn.cteria  scattered  over  the  sur- 
face, and  separated  by  £  inch  or  more.  Each  germ  thus 
isolated  from  its  neighbor  soon  multiplies  so  that  in  24  to 
36  hours  a  small  growth  of  about  the  size  of  a  pin  head 
becomes  visible.  This  isolated  growth  is  known  as  a 
colony,  and  inasmuch  as  it  is  derived  from  a  single  cell  it 
is  &pure  culture  of  that  organism. 

In  this  and  all  subsequent  work  successful  results  and 
freedom  from  danger  depend  upon  the  rigid  sterilization 
of  all  articles  used.  Hence,  sterilize  all  instruments, 
wires,  etc.,  immediately  before  use,  and  immediately  after 
use,  before  placing  them  back  on  the  table  or  in  the 
tumbler. 

LABORATORY  WORK. — Make  dilution  culture  (on  three 
potatoes)  of  the  Micrococcus  prodigiosus. 

Mass  culture  (single  potato)  of  Orange  sarcine. 
Red  bacillus  of  water. 
Violet  bacillus  of  water. 


—  10  — 


III. 

LIFE  HISTORY  OF  A  BACTERIAL  CELL. 

Young  cells  grow,  attain  full  size,  multiply. 
Spore  formation,  analogy  of  spores  to  seeds  of  higher 
plants. 

Observed  in  many  bacilli,  few  spirals,  but  not  in 

micrococci. 

Sporogenic  granules — their  coalescence — the  spore. 
Spore  germination  observed  in  but  few  instances. 

Bacillus  snbtilis,  Cohn  ;  Bacillus  butyricus,  Praz- 

movski. 
Bacillus   anthraci?,  Koch  ;  Bacillus  megaterium, 

DeBary. 

Each  spore  gives  rise  to  but  one  bacterial  cell,  and  a 
cell  develops  but  one  spore.  Spore  formation,  therefore, 
a  means  of  reproduction,  not  of  multiplication. 

Structure  of  a  spore— dense,  highly  resistant  cell- wall 
— the  contents. 

Behavior  of  aniline  dyes.     Action  of  heat,  cold, 

desiccation,  chemicals. 

Their  importance  as  resting  or  permanent  forms. 
Position  of  the  spore  in  the   cell — median  or  termi- 
nal— with   or  without  enlargment.     Clostridium  form — 
Drumstick  or  u  Kopchen  v  form. 

Attempts  at  classification  —  Endospore  and  arthro- 
spore  bacteria. 

Spore  formation  not  the  result  of  exhaustion  of  soil, 
but  like  the  flower  and  fruit  of  plants  represents  the  high- 
est stage  of  development.  It  occurs  only  under  favorable 
conditions—  medium,  temperature,  oxygen. 

Asporogenic  bacteria,  result  of  unfavorable  environ- 
ment— influence  of  calcium. 


MEMORANDA. 


MEMORANDA. 


—  11  — 

Multiplication    of  bacteria    always  takes    place    by 
division — one  cell  forms  two  and  only  two  new  cells. 

Threads — result  from  division  of  bacilli  which 
remain  adherent  end  to  end  by  the  undivided 
cell  membrane. 

Diplocoocus — Streptococcus — Staphylococcus. 
Division  in  two  directions  results  in  Tetrads. 
Division  in  three  directions  results  in  sarcines. 

Demonstration  of, 

Sporogenic  granules,  threads,  diplococci,  Staphyl- 

ococci,  streptococci,  tetrads,  sarcines. 
Spores,  median  and  terminal. 


—  12  — 


IV. 
THE  MICROSCOPE  AND  ITS  COMPONENT  PARTS. 

The  stand — Coarse  adjustment  by  rack  and  pinion. 

Fine  adjustment  by  micrometer  screw. 
The   optical  part— mirror,  Abbe   condenser  with  iris 
diaphragm,  objectives,  eye-pieces. 

(Structure  of  the  Abbe  condenser — its  object. 

In  examining  unstained  specimens  contract 

the  diaphragm. 
In   examining    stained    specimens  open  the 

diaphragm. 
Requirements  of  good  objectives  : 

Magnification,  defining  power,  resolving  power. 
Achromatic  and  apochromatic  objectives. 
Homogeneous  oil-immersion  objectives — advant- 
ages. 

The  cedar  oil  must  be  removed  at  the  close  of  the 
day's  work  by  touching  the  lens  carefully  with  soft  filter 
paper. 

Slides  and  cover-glasses  for  microscopic  work  must  be 
rigidly  clean.  The  cover-glasses  should  be  kept  in  alco- 
hol and  wiped  dry  when  needed.  If  surface  is  at  all 
greasy,  heat  the  cover-glasses  with  sulphuric  acid  and 
potassium  bichromate,  wash  well  with  water  and  transfer 
to  alcohol. 

EXAMINATION  OF  BACTERIA  IN  HANGING 

DROPS. 

This  is  intended  to  show  the  bacteria  in  the  living 
condition — their  form  and  arrangement,  presence  or 
absence  of  motion,  the  appearance  of  the  protoplasm, 
division  of  the  cells,  sporogenic  granules,  spores,  etc. 


MEMORANDA. 


MEMORANDA. 


—  13  — 

Place  a  small  drop  of  water  in  the  center  of  a  clean 
cover  glass.  With  a  sterilized  and  cooled  platinum  wire, 
which  is  fused  into  the  end  of  a  glass  rod,  6  or  7  inches 
long,  transfer  a  minute  amount  of  the  bacterial  growth 
from  the  surface  of  one  of  the  potato  cultures,  to  this  drop 
of  water.  Place  the  cover-glass  on  a  table  or  on  a 
block.  Select  a  clean  "  concave  slide  "  and  apply  a  ring 
of  vaseline  with  a  brush  or  stick  to  the  edge  of  the 
cavity.  Invert  the  slide  thus  prepared  over  the  cover- 
glass  with  the  drop  and  gently  press  down  till  the  little 
chamber  is  sealed  air  tight.  The  slide  is  now  ready  for 
microscopic  examination,  and  if  properly  made  the  drop 
of  water  will  be  flat,  and  will  not  run  when  the  slide  is 
placed  on  its  edge. 

Place  the  slide,  with  the  cover-glass  uppermost,  on 
the  stage  of  the  microscope,  .and  first  find  the  edge  of  the 
drop  with  a  low  power — No.  3  objective.  If  too  much 
light  is  present,  constrict  the  diaphragm.  The  edge  of  the 
drop  should  be  seen  as  a  sharp  line  passing  through  the 
field  of  the  microscope.  Holding  the  slide  between  the 
thumb  and  forefinger  of  the  left  hand,  slowly  move  it  so 
that  the  edge  of  the  drop  constantly  remains  in  the  field. 
In  this  way  the  entire  edge  or  circumference  of  the  drop 
should  be  examined,  chiefly  for  the  purpose  of  practice  in 
moving  a  slide  under  the  microscope.  Owing  to  the  min- 
ute size  of  bacteria  they  cannot  be  seen  under  this  mag- 
nification. To  observe  the  individual  cells,  therefore, 
recourse  must  be  had  to  the  higher  powers — No.  7  objec- 
tive, or  the  -iV  inch  homogeneous  oil-immersion  objective. 

Examination  with  No.  7  objective. — Having  found  the 
edge  of  the  drop  with  No.  3  objective,  replace  this  by  No. 
7,  by  rotating  the  nose-piece.  Then  lower  the  tube  of  the 
microscope  by  the  coarse  adjustment  till  the  objective 
almost  touches  the  cover-glass.  The  field  of  the  micro- 
scope now  is  usually  very  dark,  hence  open  the  diaphragm 
a  trifle  to  admit  enough  light  to  see  distinctly.  With  the 
fine  adjustment  now  raise  the  microscope  tube  till  the 
edge  of  the  drop  is  brought  out  distinctly.  By  focussing 


—  14  — 

the  edge  carefully  the  bacteria  will  be  readily  detected. 
Now  move  the  slide  as  mentioned  above,  so  as  to  examine 
the  entire  edge  of  the  drop,  and  also  the  center.  Study  the 
characteristics  of  the  microorganisms  present. 

In  working  with  high  powers,  while  focussing,  it  is 
desirable  to  constantly  hold  the  slide  between  the  thumb 
and  forefinger  of  the  left  hand,  imparting  to  it  a  slight 
motion.  If  this  motion  is  arrested  it  is  due  to  pressure  of 
the  objective,  which  has  been  lowered  too  far,  and  unless 
the  pressure  is  promptly  relieved,  damage  may  result. 

Examination  with  A-  homogeneous  oil  immersion 
objective. — Having  studied  the  bacteria  in  the  hanging 
drop  with  the  No.  7  objective,  replace  No.  3  objective  and 
again  find  the  edge  of  the  drop.  Now  raise  the  tube  of 
the  microscope,  bring  the  iV  objective  into  position. 
Place  a  drop  of  cedar  oil  on  the  center  of  the  cover-glass, 
and  lower  the  tube  till  the  objective  touches  the  oil.  As 
the  field  is  now  very  dark  open  the  diaphragm  slightly. 
Focus  the  edge  of  the  drop  with  the  fine  adjustment, 
holding  the  slide  between  the  fingers  of  the  left  hand. 
Examine  carefully  the  bacteria  present,  their  motion, 
structure,  etc.,  and  also  different  parts  of  the  drop  in  the 
manner  already  indicated. 

LABORATORY  WORK. — Make  hanging  drops  of  the  four 
kinds  of  bacteria  growing  on  the  potatoes,  and  examine  as 
above.  Too  much  time  cannot  be  devoted  to  the  work  at 
this  point,  as  the  practice  thus  obtained  is  indispensable 
to  the  easy  and  successful  manipulation  of  the  microscope. 


MEMORANDA. 


MEMORANDA. 


V. 

REQUIREMENTS  OF  BACTERIA. 

Bacteria,  like  all  living  cells,  require  certain  nourish- 
ing substances. 

Absence  of  chlorophyll — inability  to  acquire  carbon 
from  carbonic  acid.  Carbon,  therefore,  derived  from  pre- 
formed carbon  compounds — sugar,  proteids,  etc. 

Nitrogen  derived  from  organic  and  inorganic  sources 
--proteids,  nitrates,  nitrites,  ammonia. 

Presence  of  moisture  necessary  for  growth. 

Suitable  reaction  of  medium — neutral  or  slightly  alka- 
line. 

These  conditions — solutions  of  organic  nitrogenous 
substances — widely  distributed  in  nature.  Hence  the 
occurrence  of  bacteria  almost  everywhere  on  the  surface 
of  the  ear tli. 

Almost  total  absence  of  bacteria  at  high  altitudes,  in 
air  of  mid  ocean,  and  in  deeper  layers  of  the  earth. 

Absence  of  bacteria  in  organs  and  circulating  fluids 
of  the  healthy  normal  body. 

Theory  of  spontaneous  generation,  the  outcome  of  the 
wide  distribution  of  bacteria  and  lack  of  knowledge  con- 
cerning the  resistance  of  bacteria  and  their  spores.  Its 
overthrow. 

Classification  of  bacteria  according  to  habitat — sapro- 
phytic  and  parasitic.  No  sharp  line  of  distinction  to  be 
drawn — facultative  and  obligative. 

Temperature  requirements.  Each  organism  has  its 
minimum,  optimum  and  maximum  temperature  for 
growth. 

In  general  the   minimum  temperature  is   about 
15°  C.;  the  maximum  about  40°. 


—  16  — 

Optimum   temperature  of  saprophytic   bacteria, 

that  of  the  room,  or  summer,  25°  to  30°  C. 
Optimum  temperature  of  parasitic  bacteria,  that  of 

the  body,  about  37.5°  0. 
Influence  of  cold — of  heat,  70°  and  above. 
Growth  of  some  bacteria  at  0°.    At  50°,  60°,  70°  C. 
Injurious  action  of  diffuse   light,   and   especially  of 
sunlight. 

LABORATORY  WORK. — Continuation  of  that  of  preceding 
day. 


MEMORANDA. 


MEMORANDA. 


VI. 
CHEMISTRY  OF  BACTERIA. 

All  living  cells  take  in  food,  elaborate  certain  pro- 
ducts and  give  off  waste  or  metabolic  products. 
Production  of, 

Gases — Carbonic  acid,  hydrogen  sulphide,  nitro- 
gen, etc. 

Acids — Acetic,  lactic,  butyric,  phenyl-propionic, 
etc.  Amido  acids.  Nitrous  and  nitric  acids. 

Alkalies — Ammonia,  and  its  substitution  com- 
pounds, the  amines. 

Ptomaines — Alkaloidal  compounds  like  the  vege- 
table alkaloids.  May  be  poisonous  (toxines) 
and  non-poisonous. 

Proteids  —  Bacterial  proteids  may  be  highly 
poisonous.  Compare  with  the  phytalbumoses 
of  plants,  and  the  proteids  in  venom  of  ser- 
pents. 

Soluble  ferments  or  enzymes — Analogy  to  the 
soluble  ferments  of  the  animal  body  and  of 
certain  plants. 

Alcohols — Ethyl,  propyl,  butyl;  phenol,  etc. 
Reducing  powers — oxidizing  powers. 

Classification  of  bacteria  according  to  function. 
Zymogenic — fermentation  bacteria. 
Saprogenic — putrefaction  bacteria. 
Chromogenic — pigment  producing  bacteria. 
A  erogenic — gas  producing  bacteria. 
Photogenic — light  producing  or  phosphorescing 

bacteria. 

Fermentations    as  vital   phenomena,   the    result    of 
activity  of  microorganisms,  so-called  organized  ferments, 


—  18  — 

bacteria,  yeasts,  moulds,  etc. ;  unorganized  or  soluble  fer- 
ments (enzymes)  produced  by  bacteria. 

Alcoholic,  acetic,  butyric  acid,  etc.,  fermentations. 

Ferment  changes  in  the  mouth — dental  caries. 

Abnormal  fermentation  in  the  stomach — in  the  intes- 
tines, Summer  diarrhoea  of  infants. 

Ammoniacal  fermentation  of  urine — Hydrothionuria. 

Putrefaction  is  putrid  fermentation — Proteids  acted 
upon  whereas  in  true  fermentations,  starches,  sugars,  and 
cellulose  are  acted  upon. 

Poisonous  foods. 

Liquefaction  of  albumin,  gelatin,  etc.,  by  bacteria  due 
to  soluble  peptonizing  ferments. 

Liquefying  and  non-liquefying  bacteria. 

Inversion  of  starch  by  bacteria — coagulation  of  milk. 

Nitrification  in  the  soil. 

Production  of  pigment,  as  a  rule,  is  a  secondary  process, 
taking  place  outside  of  the  cell.  The  oxygen  of  the  air 
acts  on  a  colorless  or  leuco-product. 

Sometimes  the  pigment  may  be  formed  directly  by 
the  cell — primary  product. 

Phosphorescence — the  result  of  intracellular  activities. 

In  fermentation  and  putrefaction  more  or  less  com- 
plex, dead  animal  and  vegetable  substances  are  acted 
upon  by  microorganisms;  transformed  into  relatively  simp- 
ler compounds,  and  eventually  into  inorganic  forms,  as 
carbonic  acid,  ammonia,  nitrates,  nitrites,  etc.,  which  are 
now  utilizable  by  living  plants.  Thus,  lifeless  remains 
become  indispensable  to  new  life,  and  bacteria,  in  their 
role  of  scavengers  of  nature,  prove  beneficial. 

Certain  bacteria  may  live  on  living  matter  in  the  ani- 
mal body,  and  in  plants,  not  only  at  their  expense,  but 
even  to  their  injury,  producing  changes  which  may  result 
in  disease  and  death. 

Division  of  bacteria  into  pathogenic  and  non-patho- 
genic,—toxicogenic  and  non-toxicogenic. 


MEMORANDA. 


MEMORANDA, 


l/U 


—  19  — 

STAINING  OF  BACTERIA. 

Aniline  dyes  are  commonly  employed  for  this  purpose, 
and  those  which  are  used  most  frequently  are  fuchsine, 
gentian  violet,  methyl  violet,  methylene  blue,  and  vesuvin 
or  Bismarck  brown.  The  first  two  are  quite  permanent, 
stain  rapidly  and  deeply  and  as  a  rule  are  to  be  preferred. 
Methylene  blue  stains  slowly  and  is  excellent  for  special 
purposes. 

Saturated  solutions  in  absolute  or  strong  alcohol  are 
first  prepared  and  serve  as  stock.  These  concentrated 
solutions  are  only  very  rarely  used  as  such.  Ordinarily 
they  are  diluted  with  water.  For  this  purpose  pour  into 
the  small  staining  bottle  (1  oz.),  which  is  provided  with  a 
pipette,  some  of  the  concentrated  solution  to  a  depth  of 
about  i  inch,  then  fill  the  bottle  with  water.  This  dilute 
stain  must  be  perfectly  clear  from  cloud  or  precipitate, 
and  should  not  be  transparent.  The  dilute  stains  do 
not  keep  for  any  length  of  time  owing  to  the  deposit  of 
the  dye.  In  such  cases  a  fresh  solution  should  be  made. 

SIMPLE  STAINING  OF  COYER-GLASS  PREPARATIONS. 

Place  a  small  drop  of  water  on  a  cover-glass  which  is 
held  between  the  thumb  and  forefinger  of  the  left  hand. 
The  cover-glass  must  be  perfectly  clean  so  that  when  the 
drop  is  subsequently  spread  over  the  surface  with  a  plat- 
inum wire,  it  will  spread  in  a  thin  film  and  not  gather  into 
minute  globules.  The  drop  of  water  taken  should  prefer- 
ably be  so  small  that  when  spread  in  a  thin  film  over  the 
cover-glass,  it  will  dry  almost  immediately,  and  thus 
evenly  cover  the  whole  surface.  Large  drops  of  water  by 
drying  slowly,  tend  to  leave  unsightly  "  shore-lines." 

With  a  sterilized  platinum  wire  pick  up  a  minute 
amount  of  the  growth  from  one  of  the  potato  cultures  and 
touch  the  drop  of  water  cautiously  once  or  twice.  Then 
sterilize  the  wire,  and  when  cool,  spread  the  drop  of  water 
evenly  over  the  whole  surface  of  the  cover  glass,  avoiding, 
however,  contact  with  the  fingers.  In  a  few  minutes  the 
water  evaporates,  and  if  desired,  this  may  be  hastened  by 
3 


—  20  — 

waving  the  cover  glass,  to  and  fro,  over  a  flame,  at  a  height 
of  6  or  8  inches.  Care  must  be  taken  not  to  transfer  too 
much  material  to  the  drop  of  water,  for  in  such  cases  the 
cover-glass  on  subsequent  staining  will  be  found  to  be  one 
mass  of  bacteria. 

The  cover-glass  prepartion  which  has  now  been  dried 
in  the  air  or  over  a  flame,  must  be  u  fixed"  before  apply- 
ing the  staining  solution.  For  this  purpose,  take  hold  of 
the  cover-glass  with  a  narrow-pointed  pair  of  forceps, 
and  pass  it  rapidly  through  the  flame,  from  above  down- 
ward, keeping  the  specimen  side  up,  away  from  direct 
contact  with  the  flame.  If  the  exposure  to  the  flame  is 
too  long  the  bacteria  may  be  so  altered  as  to  refuse  to  take 
the  stain  subsequently,  or  at  most  poorly.  On  the  other 
hand,  if  not  heated  enough,  the  bacteria  will  readily  wash 
away  on  treatment  with  the  dye,  or  with  water. 

As  soon  as  the  fixed  cover-glass  is  cool,  and  while  still 
holding  it  in  the  forceps,  cover  the  specimens  with  the 
dilute  aniline  stain.  Allow  this  to  act  for  the  necessary 
length  of  time  (i — ^— 1  min.)  and  then  wash  off  com- 
pletely by  means  of  a  syphon  or  bulb  wash-bottle.  Place 
the  cover-glass  now  on  a  piece  of  filter-paper,  resting  on 
the  right  index  finger,  and  rotate  carefully  till  the  lower 
side  is  perfectly  clean  and  dry.  Now  invert  the  specimen, 
with  the  moist  specimen  side  downward,  onto  a  clean  glass 
slide.  Sufficient  water  should  be  present  to  fill  in  the 
space  between  the  cover-glass  and  slide.  Specimens 
should  never  be  examined  dry. 

Place  the  slide  thus  prepared  on  the  stage  of  the 
microscope  and  first  examine  with  the  No.  7  objective  and 
subsequently  with  the  ^  homogeneous  oil-immersion 
objective,  in  the  same  way  as  was  done  in  examining 
hanging  drops. 

A  good  cover-glass  should  show  the  bacteria  well 
stained,  not  in  masses,  but  separated  from  each  other,  and 
evenly  distributed  over  the  entire  cover-glass.  If  the 
stained  bacteria  are  seen  to  move  about  it  is  due  to  insuf- 
ficient fixing  in  the  flame. 


MEMORANDA. 


—  21  — 

LABORATORY  WORK.  —  Practice  staining  cover-glass 
preparations  made  from  the  different  potato  cultures,  em- 
ploying the  five  aniline  dyes  mentioned  above.  Also 
stain  preparations  made  from  the  white  matter  on  the 
teeth,  near  the  edge  of  the  gums,  and  look  for  comma 
bacilli,  spirilla,  and  leptothrix  threads. 

To  make  permanent  stained  preparations,  the  speci- 
men which  has  proven  satisfactory  on  preliminary  exam- 
ination in  water  as  above,  can  be  floated  off  the  slide  by 
first  bringing  a  drop  or  two  of  water  near  the  edge  of  the 
cover-glass.  If  any  oil  is  on  the  upper  side  it  should  be 
carefully  removed  by  rotating  the  cover-glass  on  a  piece 
of  filter-paper.  The  specimen  is  then  allowed  to  dry  in 
the  air,  or  by  gently  waving  over  aflame.  A  clean  glass 
slide  is  then  selected  and  a  suitable  drop  of  Canada  bal- 
sam placed  in  the  center.  The  dry  cover-glass  is  then 
inverted,  specimen  side  down,  and  carefully  lowered  until 
it  touches  the  balsam.  If  necessary,  gentle  pressure  is 
applied  so  as  to  cause  the  balsam  to  spread  out  under  the 
cover-glass. 

The  following  synopsis  will  be  of  service : 

Simple  Stain. — Cover-glass  preparation. 
Air- dried. 
3  x  through  flame. 
Dilute  stain  (^ — 1  min.). 
Water  (and  examine). 
Air-dried. 
Canada  balsam. 

GELATIN  PLATE  CULTURE. 

Tha-  object  of  this  method,  as  with  the  dilution  potato 
culture  already  made,  is  to  isolate  the  several  kinds  of 
bacteria  that  may  be  present.  The  isolated  organisms 
developing  in  a  solid,  transparent  medium,  form  colonies 
which  are  easily  perceived  and  from  which  transplan- 
tations can  be  readily  made.  Pure  cultures  of  the  differ- 
ent kinds  of  bacteria 'are  thus  obtained. 


—  22  — 

First,  sterilize  six  glass  plates  by  placing  them  in  an 
iron  box  and  heating  this  in  the  dry  heat  sterilizer,  at  a 
temperature  of  150-175°  0.,  for  one  hour.  Then  remove  the 
box  and  allow  it  to  cool. 

PJace  three  of  the  sterilized  gelatin  tubes  in  a  water- 
bath  which  has  been  warmed  to  about  30-35°  0.  When 
the  gelatin  melts  the  tubes  are  ready  for  inoculation. 
With  a  sterilized,  cooled  platinum  wire  pick  up  a  minute 
amount  of  the  growth  of  the  potato  culture  of  Micrococcus 
prodigiosus.  Place  one  of  the  liquefied  gelatin  tubes 
between  the  thumb  and  index  finger  of  the  left  hand,  so 
that  it  is  almost  horizontal.  The  neck  of  the  tube  with 
its  plug,  as  well  as  the  palm  of  the  left  hand,  is  turned  to 
the  right.  While  still  holding  the  platinum  wire  in  the 
right  hand,  grasp  the  cotton  plug  with  the  little  finger  of 
that  hand,  and  remove  it  by  slight  rotation.  Now  pass 
the  inoculated  wire  into  the  tube  and  thoroughly  mix  the 
bacteria,  thus  introduced,  with  the  gelatin.  Then  with- 
draw the  wire,  replace  the  cotton  plug,  and  sterilize  the 
platinum  wire  in  a  flame. 

With  a  colored  wax  pencil  mark  the  tube  thus  inocu- 
lated with  J.  Likewise  mark  another  liquefied  gelatin 
tube  with  2.  Place  tube  1  in  the  left  hand  in  the  same 
position  as  before,  and  then  next  to  it,  tube  2.  Remove 
the  cotton  plug  of  tube  2  and  place  it  between  the  adjoin- 
ing index  and  middle  fingers.  Then  remove  the  cotton 
plug  of  tube  1  and  place  it  between  the  ring  and  little 
finger.  Now,  with  a  sterilized  cooled  platinum  wire,  the 
end  of  which  is  provided  with  a  small  loop,  transfer  a 
loopful  of  gelatin  from  tube  1  to  tube  2  and  mix  well. 
Return  the  platinum  wire  to  tube  1  and  again  transfer  a 
loopful  of  gelatin  to  tube  2.  Repeat  this  once  more,  so 
that  all  told,  three  transfers  of  inoculated  gelatin  have 
been  made.  Replace  the  cotton  plugs  into  their  respect- 
ive tubes,  sterilize  the  platinum  wire  and  set  the  tubes  in 
a  tumbler  having  a  layer  of  cotton  on  the  bottom. 

Mark  a  new  liquefied  gelatin  tube  with  3.  Then  place 
2  in  the  same  position  in  which  No.  1  was  just  held,  and 


MEMORANDA. 


MEMORANDA, 


—  23  — 

next  to  it  place  tube  3.  Remove  the  cotton  plugs  and 
place  in  their  respective  places  as  before.  With  the  ster- 
ilized cool  platinum  wire  make  three  successive  transfers 
of  gelatin  from  tube  2  to  tube  3.  Return  the  cotton 
plugs  to  their  tubes,  sterilize  the  wire,  and  set  the  tubes 
aside  in  the  tumbler. 

Each  of  the  three  gelatin  tubes  has  now  been  inocu- 
lated. Tube  1  usually  has  a  very  large  number  of  bac- 
teria, while  tube  2  has  less  and  tube  3  should  have  but  a 
small  number,  so  that  subsequently  when  colonies  develop 
these  should  be  separated  from  one  another  by  an  appre- 
ciable distance.  It  is  necessary  therefore,  to  take  a  very 
minute  amount  of  material  for  the  inoculation  of  tube  1 
in  order  to  obtain  good  dilutions.  In  transferring  gelatin 
from  one  tube  to  another  care  must  be  taken  to  prevent 
the  wire  from  coming  in  contact  with  the  neck  or  walls  of 
the  tubes. 

Prepare  the  ice  plating  apparatus  for  use  and  then 
level  it.  Remove  a  sterilized  glass  plate  from  the  iron 
box  by  grasping  the  edges  with  two  fingers ;  place  it  upon 
the  ground  plate  of  the  ice  apparatus  and  cover  with  the 
bell  jar.  As  soon  as  the  plate  is  cool  it  is  ready  to  receive 
the  gelatin.  Before  pouring  the  contents  of  the  tubes 
upon  the  plates  it  is  necessary,  as  a  matter  of  precaution, 
to  sterilize  the  neckof  the  tube.  To  accomplish  this,  cut 
off  the  cotton  which  projects  from  the  tube,  push  in  the 
plug  a  trifle,  and  then  rotate  the  neck  of  the  tube  in  a 
flame  till  the  cotton  begins  to  turn  yellow.  As  soon  as 
the  neck  oi  the  tube  cools  the  gelatin  can  be  poured.  To 
do  this  remove  the  cotton  plug  with  a  pair  of  forceps,  ster- 
ilized in  the  flame,  and  place  it  between  the  fingers  of  the 
left  hand.  Transfer  the  tube  to  the  right  hand,  raise  the 
bell  jar  somewhat  and  pour  the  gelatin  onto  the  centre  of 
the  plate.  With  the  lip  of  the  tube  spread  out  the  gela- 
tin as  rapidly  and  as  fully  as  possible,  avoiding,  however, 
the  edges  of  the  plate.  Allow  the  gelatin  to  solidify 
under  cover  of  the  bell-jar. 


—  24  — 

Prepare  a  "  moist  chamber"  in  the  same  way  as  for 
potato  cultures  except  that  tap  water  may  be  used  for 
moistening  instead  of  mercuric  chloride.  On  three  small 
pieces  of  paper  write  the  name  of  the  germ  or  material, 
the  number  of  the  plate  and  date.  Now  place  a  glass  bench 
on  the  bottom  of  the  moist  chamber  and  on  it  the  label 
for  plate  1.  Transfer  the  gelatin  plate  from  the  ice  appa- 
ratus to  the  bench.  Pour  the  remaining  gelatin  tubes  on 
plates  in  the  same  manner  as  described  and  when  cool 
transfer  to  the  benches  which  are  arranged  one  above  the 
other,  in  the  moist  chamber.  Each  chamber  can  hold  a 
stack  of  six  plates. 

The  moist  chamber  containing  the  plates  is  now  set 
aside  for  1-2-3  days,  during  which  time  colonies  will 
develop  and  be  ready  for  further  examination. 

LABORATORY  WORK.— Make  plate  cultures  of  Micrococ- 
cus  prodigiosus  and  of  Bacillus  Indicus. 


MEMORANDA. 


MEMORANDA. 


—  25  — 


YJII. 

EXAMINATION  OF  COLONIES. 

In  a  greater  or  less  period  of  time,  varying  usually 
from  1  to  3  days,  colonies  develop  on  the  plates,  and  as 
soon  as  they  become  of  suitable  size  they  are  ready  for 
examination.  A  careful  study  of  the  colonies  on  a  plate 
should  first  be  made  with  the  unaided  eye.  Several  char- 
acteristics of  growth  can  often  thus  be  recognized  quite 
early.  Especial  attention  should  be  given  to  the  form 
and  appearance  of  the  colonies;  the  presence  or  absence 
of  pigment;  liquefaction  or  non-liquefaction  of  gelatin, 
etc.  It  should  be  remembered,  however,  that  a  given 
organism  may  give  rise  to  at  least  two  kinds  of  colonies 
which  sometimes  are  quite  different  in  appearance.  Thus, 
we  may  have  surface  colonies,  and  also  deep  colonies. 
The  former  developing  on  the  surface  of  the  gelatin  are 
unhindered  in  their  development,  and  may,  therefore, 
spread  out  and  thus  acquire  peculiar  characteristics,  more- 
over, having  ready  access  to  oxygen,  pigment  formation 
and  liquefaction  will  be  first  seen  in  connection  with  these 
surface  colonies.  The  deep  colonies,  on  the  other  hand, 
are  surrounded  on  all  sides  by  solid  gelatin,  and  hence, 
much  the  same  resistance  to  growth  will  exist  in  all  direc- 
tions. The  result  is  that  deep  colonies,  as  a  rule,  are 
much  alike  in  appearance.  Thus,  the  form  is  usually 
round  or  oval,  with  sharp  edges,  and  the  contents  are 
slightly  granular  and  yellowish. 

The  plate  should  now  be  placed  upon  the  stage  of  the 
microscope  and  the  colonies  carefully  examined  with  a 
low-power — No.  3  objective.  Further  characteristics  can 
thus  be  brought  out  which  have  escaped  the  eye. 

The  study  of  the  micro-organisms  which  compose  the 


—  26  — 

colonies  should  now  be  made.  This  is  done  by  making 
hanging  drop  examinations  and  stained  preparation  accord- 
ing to  the  directions  already  given. 

The  object  in  making  plate  cultures  is  to  obtain  colo- 
nies which,  since  they  are  derived  from  a  single  cell,  are 
pure  cultures  of  that  organism.  To  perpetuate  and  keep 
up  the  pure  culture  thus  obtained,  it  is  necessary  to  resort 
to  transplantation.  For  this  purpose  the  colony  to  be 
transplanted  is  touched  with  a  sterilized  and  cooled, 
straight  platinum  wire.  A  portion  of  the  colony  will 
adhere  to  the  end  of  the  wire  and  can  be  transferred 
to  a  tube  of  sterilized  gelatin.  The  wire  is  usually  pushed 
down  the  centre  of  the  tube,  in  which  case  we  have  what 
is  known  as  a  stich  or  stab  culture.  The  operation  of 
touching  the  colony  is  one  that  requires  the  greatest  care 
to  prevent  contamination  with  foreign  colonies,  or  other 
material,  thus  vitiating  the  pure  culture.  For  this  reason 
it  is  always  carried  out  under  a  microscope,  and  so  far  as 
patience  is  concerned  it  certainly  is  not  inaptly  called 
"fishing." 

The  gelatin  plate  is  placed  on  the  stage  of  the  micro- 
scope and,  with  the  No.  3  objective,  a  suitable  colony  for 
transplantation  is  selected.  It  is  desirable  to  have  but  one 
colony  in  the  field  of  the  microscope.  A  straight  platinum 
wire,  previously  sterilized  and  cooled,  is  held  in  the  right 
hand  in  the  pen  position.  The  hand  is  supported  by  rest- 
ing the  little  finger  on  the  right  corner  of  the  stage.  The 
platinum  wire  is  then  inserted  about  midway  between  the 
front  lens  of  the  objective  and  the  surface  of  the  gelatin. 
It  is  held  steadily  in  this  position,  and  on  looking  into  the 
microscope  an  indistinct  shadow  is  seen.  The  wire  is 
slowly  drawn  back  till  the  end  of  the  shadow  or  indistinct 
wire  is  directly  over  the  colony.  Should  the  wire  in  doing 
this  touch  the  objective  or  the  gelatin  it  must  be  sterilized 
at  once  and  the  operation  repeated.  When  the  end  of  the 
wire  has  been  brought  over  the  colony,  gradually  lower 
the  point  till  it  touches  the  colony  or  cuts  it  into  two. 


MEMORANDA. 


MEMORANDA. 


—  27  — 

Now  carefully  remove  the  wire  without  touching  the 
microscope  or  some  other  portion  of  the  gelatin.  A  tube 
of  solid  gelatin  is  held  in  the  left  hand  in  an  almost  hori- 
zontal position,  the  plug  is  then  removed  by  grasping  it 
with  the  little  finger  of  the  right  hand,  and  the  platinum 
wire,  which  has  a  small  portion  of  the  colony  attached  to 
it,  is  slowly  forced  down  the  centre  of  the  gelatin  to  the 
bottom  of  the  tube.  The  cotton  plug  is  at  once  replaced, 
the  wire  sterilized  and  the  tube  set  aside. 

The  stich  culture  thus  made  is  a  pure  culture  and  is 
now  labelled  with  the  name  of  the  organism  and  date  and 
set  aside  to  develop.  In  a  few  days  development  takes 
place  along  the  line  of  inoculation  and  more  or  less  char- 
acteristic growth  results. 

The  manner  of  growth  should  be  daily  observed. 
Hanging-drop  and  stained  preparations  can,  of  course,  be 
made  if  desired,  from  tube  cultures.  When  the  gelatin 
is  very  old,  and  hence  too  solid,  it  tends  to  split  as  soon  as 
the  platinum  wire  is  forced  into  it.  This  is  remedied  by 
melting  the  gelatin  and  allowing  it  to  re-solidify. 

LABORATORY  WORK. — Examine  carefully  the  colonies 
with  the  eye  and  under  the  microscope.  Make  hanging- 
drop  and  stained  preparations  from  the  colonies.  Prac- 
tice "fishing"  and  make  stich  cultures  from  each  of  the- 
different  colonies. 

The  line  of  study  of  the  Micrococcus  prodigiosus  and 
Bacillus  Indicus  and  of  the  various  bacteria  to  be  pres- 
ently taken  up,  consists  first,  in  making  plate  cultures. 
Colonies  are  thus  obtained,  the  characteristics  of  which 
are  to  be  thoroughly  studied.  Hanging-drop  examination! 
and  stained  preparation  are  next  made,  thus  becoming; 
familiar  with  the  organism  itself.  Stich  cultures  in  gela 
tin  are  then  made  and  also  streak  cultures  on  potato  or 
agar  which  will  be  presently  described.  Finally  drawings 
should  be  made  showing  the  form  of  the  colony,  the  form 
of  the  organism,  the  appearance  of  the  stich  culture,  etc. 

4 


—  28  — 

Summarized  then,  each  organism,  subsequently  de- 
scribed, is  to  be  studied  by  making — 
Plates, 

Colonies, 

Hanging-drop  examination. 
Stained  preparation, 
Stich  culture  in  gelatin, 
Streak  culture  on  potato,  or  agar,  or  both. 
Drawings. 


MEMORANDA. 


MEMORANDA. 


—  29  — 


IX. 
MODIFIED  GELATIN  PLATE  CULTURES. 

Several  modifications  of  the  Koch  plate  method  as 
just  described  have  been  introduced  whereby  the  same,  if 
not  better  results  are  obtained  with  less  apparatus.  In 
the  plate  method  contamination  not  infrequently  results 
from  exposure  to  the  air  while  on  the  ice  apparatus,  or 
subsequently  when  kept  in  the  large,  moist  chamber.  An 
examination  of  one  plate  necessitates  the  exposure  of  the 
remaining  plates  to  contamination  with  the  organisms  in 
the  air.  Furthermore,  it  not  infrequently  happens  that 
the  gelatin  on  an  upper  plate  undergoes  liquefaction  and 
then  drips  over  the  edges  of  the  plates  on  those  below  it. 
To  overcome  these  difficulties  Petri  introduced  the  use  of 
shallow  dishes  which  are  about  10  cm.  in  diameter. 

Petri  Dish  Culture. — Place  the  Petri  dishes  in  a  wire 
basket  and  sterilize  in  the  diy-heat  oven  by  heating  I 
hour  at  a  temperature  of  150-175°  C.,  then  allow  to  cool. 
Inoculate  three  gelatin  tubes  with  the  organism  to  be 
plated  in  the  same  manner  as  for  ordinary  plates.  Cut  off 
the  projecting  cotton,  sterilize  the  lip  of  the  tube  as  before, 
then  pour  the  contents  of  each  tube  into  one  of  the  cool, 
sterilized  Petri  dishes,  properly  labeled.  Replace  the 
cover  and  gently  tilt  the  dish  from  side  to  side  so  as  to 
cause  the  gelatin  to  spread  evenly  over  the  bottom.  Allow 
the  gelatin  to  solidify,  then  set  the  dishes  aside  for  colo- 
nies to  develop.  When  the  colonies  develop  examine  on 
the  stage  of  the  microscope  and  transplant  as  with  ordi- 
nary plates. 

In  this  method  each  dish  constitutes  a  plate  by  itself. 
It  can  be  readily  examined  and  the  risk  of  contamination 
is  reduced  to  a  minimum.  In  addition  to  that  the  use  of 


—  30  — 

the  ice-machine,  plates,  benches,  plate  boxes,  etc.,  is  done 
away  with. 

Esmarck  Roll- Tube  Cultures. — In  this  method  the 
advantages  of  the  plate  method  are  secured  without  the 
use  of  any  extra  apparatus,  as  plates  or  dishes.  The 
inoculated  gelatin  instead  of  being  poured  out  onto  steril- 
ized plates  or  into  dishes  is  solidified  in  a  thin  film  on  the 
inside  wall  of  the  test-tube.  Another  advantage  of  this 
method  is  that  it  is  well  adapted  for  those  organisms 
which  grow  very  slowly,  and  require  a  week  or  two  to 
form  distinct  colonies.  Desiccation  of  the  gelatin  can  be 
readily  prevented  in  the  roll- tube,  whereas  it  is  much 
more  difficult  in  plate  or  dish  cultures. 

Inoculate  three  gelatin  tubes  with  the  material  to  be 
plated,  in  the  usual  manner.  Then  cut  off  the  cotton 
plug  on  each  tube  and  cover  the  end  with  a  rubber  cap. 
Place  the  tube  in  a  horizontal  position,  or  nearly  so,  in  a 
dish  of  cold,  or  ice-water  and  rotate  it  carefully  until  the 
gelatin  solidifies  in  an  even  thin  layer  over  the  inside  wall 
of  the  tube.  Avoid  contact  of  the  gelatin  with  the  cotton 
plug.  Now  set  aside  in  a  cool  place  to  develop  and  then 
examine  the  colonies  under  a  microscope  and  make  trans- 
plantations. The  operation  of  fishing  in  this  case  will,  of 
course,  require  special  care. 

LABORATORY  WORK.  —  Make  Esmarch  roll-tubes  of 
saliva  (1  loopful);  and  of  the  Violet  bacillus  of  water. 

Make  Petri  dishes  of  the  Red  bacillus  of  water. 

MODIFIED  POTATO  CULTURES. 

The  method  of  making  potato  cultures  of  bacteria  is 
open  to  much  the  same  objections  as  the  ordinary  gelatin 
plate  method.  Two  modifications,  analogous  to  the  two 
modified  gelatin  plate  cultures,  are  commonly  employed 
and  are  excellently  adapted  for  their  purpose. 
.  .  Esmarch  Potato  Cultures.— The  Esmarch  dishes, 
which  are  about  6  cm.  in  diameter  and  2cm.  high,  are  ster- 
ilized in  the  dry  heat  oven  in  the  usual  manner,  and  then 
allowed  to  cool.  A  small,  sound  potato  is  selected  and 


MEMORANDA. 


MEMORANDA. 


rcKSIl 

—  31  — 

held  with  the  thumb  and  forefinger  of  the  left  hand. 
With  a  potato  knife  held  vertically,  the  outer  edge  of  the 
potato  is  pared  circularly.  Two  horizontal  sections,  one 
upper  and  a  lower  one,  are  now  made  and  the  clean, 
sound  core  of  the  potato  thus  obtained  is  slipped  into  a 
sterilized  Esmarch  dish.  Each  dish  is  thus  supplied  with 
a  clean  potato  section.  The  dishes  are  then  placed  in  a 
steam  sterilizer  and  steamed  for  f  to  1  hour.  The  pota- 
toes will  then  be  sterilized  and  cooked. 

The  inoculation  of  the  cold  sterilized  potato  is  made 
in  essentially  the  same  way  as  in  the  ordinary  potato 
culture?.  As  each  potato  is  in  a  small  sterilized  dish  by 
itself  the  risk  of  contamination  is  very  small  under  care- 
ful and  rapid  manipulation. 

LABORATORY  WORK. — Make  dilution  cultures  of  Micro- 
coccus  prodigiosus,  using  three  of  the  Esmarch  potato 
dishes. 

Test-Tube  Potato  Cultures. — These  were  introduced 
independently  and  almost  simultaneously  by  Bolton  of 
this  country,  Globig,  of  Germany  and  Roux  of  France. 
In  convenience  and  reliability  of  cultures,  the  method 
leaves  nothing  to  be  desired. 

Clean,  plug  and  sterilize  about  6  or  8  large  test-tubes 
(|  x  (>  inches).  Also  clean  several  large  potatoes  and 
place  them  in  boiling  water  or  in  steam  sterilizer  for  about 
j  of  an  hour.  By  means  of  a  cork-borer,  having  nearly 
the  diameter  of  the  test  tubes,  punch  out  a  number  of 
cylinders  from  the  cooled  potatoes.  Place  these  cylinders 
on  a  clean  piece  of  paper,  trim  off  the  ends  and  then  cut 
each  cylinder  diagonally  into  two  pieces.  Into  each  of 
the  sterilized  test-tubes  place  one  of  these  pieces  of  pota- 
toes with  the  circular  end  lowermost.  Each  tube  then 
contains  a  piece  of  potato  having  an  inclined  surface. 
Now  sterilize  again  in  a  steam  sterilizer  for  about  f  to  1 
hour. 

Instead  of  a  cork  borer  an  excellent  substitute  may 
be  obtained  by  cutting  a  test  tube  in  two.  By  making 
cylinders  from  previously  boiled  potatoes,  the  pieces  in 


—  32  — 

the  tubes  remain  perfectly  white,  whereas  cylinders  made 
direct  from  the  raw  potato  will  frequently  become  discol- 
ored by  the  subsequent  heating. 

The  inoculation  of  the  sterilized  potato  tubes  is  easily 
done.  If  it  is  desired  to  obtain  dilution  cultures,  that  is, 
colonies,  this  can  best  be  accomplished  by  making  several 
parallel  streaks  on  the  surface  of  the  potato  with  the  end 
of  the  platinum  wire,  if  necessary  repeating  the  inocula- 
tion in  two  or  three  tubes,  using  the  same  wire. 

When  transplanting  a  pure  culture,  as  a  portion  of  a 
colony,  a  single  streak  should  be  made  along  the  middle 
of  the  inclined  potato. 

LABORATORY  WORK. — Make^streak  cultures  from  the 
colonies  of  the  bacteria  which  have  been  studiedthus  far. 


MEMORANDA. 


—  34  — 
BACILLUS  PRODIGIOSUS. 

MONAS    PRODIGIOSA   OF    EHRENBERG.        MICROCOCCUS    PRODIGIOSUS 
OF    OLDER   WRITERS. 

Origin. — Found  on  starchy  substances,  rice,  potatoes, 
moist-bread  ;  also  on  meat,  albumin,  milk,  etc.  May  form 
at  times  local  epidemics,  infecting  foods  as  bread,  meat, 
sausages,  with  production  of  pink  or  red  color.  "  Bleed- 
ing" bread  or  wafers. 

Form. — Short  rod,  slightly  longer  than  its  width. 
May  form  short  threads,  especially  in  slightly  acid  media. 
Usually  sinirle  or  in  pairs. 

Motility. — Ordinarily  shows  no  motion,  except  a 
marked  Brownian  movement.  In  acid  or  very  dilute  media 
appears  to  have  slight  motion. 

Sporulation. — Has  not  been  observed.  Possesses 
marked  resistance  to  desiccation. 

Anilin  Dyes.— Stain  readily. 

Growth. — Very  rapid. 

Gelatin  Plates.— Deep  colonies,  round  or  oval,  with  sharp  border  and 
light  brown  color.  Surface  colonies  irregular,  rough  border,  granular,  with 
reddish  centre,  and  surrounded  by  clear,  liquefied  gelatin. 

Stich  Cultures.— Rapid,  funnel-shaped  liquefaction,  extending  along 
entire  line  of  inoculation.  Red  scum  forms  on  surface  of  liquid,  eventually 
settles  and  entire  contents  of  tube  colored  bright  red. 

Streak  Cultures.— On  agar,  forms  abundant,  moist,  spreading  growth, 
having  an  intense  red  color  which  is  non-diffusible.  On  potatoes,  especially 
rapid,  slimy  growth,  with  marked  pigment  production.  The  pigment,  when 
old,  has  a  metallic  fuchsine-like  lustre.  Odor  of  trimethylamine.  On  blood 
serum,  growth  as  on  agar,  with  liquefaction. 

Milk, — Growth  takes  place  and  the  pigment  is  held  in  solution  by  the  fat 
globules. 

Oxygen  requirements. — Is  a  facultative  anaerobe. 
Pigment  is  formed  only  in  presence  of  oxygen. 

Temperature.— Grows  best  at  ordinary  room  tem- 
perature. In  incubator  ceases  to  form  pigment,  and  may 
temporarily  lose  this  property,  i.  e.,  becomes  attenuated. 

Behavior  to  Gelatin.— Rapidly  liqueties  as  result 
of  formation  of  soluble  ferment.  This  liquefying  property 
may  be  diminished  or  temporarily  lost  by  growth  in  acid 
media. 

Aerogenesis.— Strong  odor  of  trimethylamine  on 
potatoes. 

Pathogenesis. — No  pathogenic  power.  Its  soluble 
products  in  large  amounts  may  have  a  toxic  action.  The 
cellular  proteids  may  induce  suppuration.  Animals  in- 
susceptible to  malignant  oedema  are  rendered  susceptible 
by  injection  of  B.  prodigiosus.  Rabbits  inoculated  with 
anthrax  are  saved  by  injection  of  B.  prodigiosus. 


MEMORANDA. 


—  36  — 


BACILLUS  INDICUS.     Koch. 

Origin.— Isolated  in  India  from  the  contents  of  the 
stomach  of  a  monkey. 

Form. — Small,  narrow,  very  short  rod  with  rounded 
ends. 

Motility.— Actively  motile. 

Sporulation. — Not  definitely  observed. 

Anilin  Dyes. — Readily  stain. 

Growth.— Is  rapid. 

Gelatin  Plates. — Deep  colonies  are  yellowish,  with  wavy  contour.  Sur- 
face colonies  grayish  yellow,  finely  granular,  with  fibrillated  borders.  Show 
movement  of  contents,  rapidly  liquefy  and  may  show  a  light  pink  color. 

Stich  Cultures.—  Rapid  liquefaction  along  line  of  inoculation.  Dense 
flocculent  growth  settles  on  the  bottom,  and  is  grayish  or  light  pink  in  color. 
A  delicate  scum  forms  on  the  surface  and  is  colored  from  a  light  pink  to 
brick  red. 

Streak  Cultures.— On  agar,  forms  a  low,  moist,  spreading  growth,  which 
usually  is  faint  pink  in  color.  On  potatoes,  the  growth  is  low,  not  slimy  as  M. 
prodigiosus,and  the  color  is  more  marked  than  on  other  media.  On  blood 
serum,  liquefaction  results  with  or  without  pigment  production. 

Oxygen  requirements. — Grows  best  in  the  pres- 
ence of  air,  but  is  a  facultative  anaerobe.  Pigment  pro- 
duction depends  upon  the  presence  of  oxygen. 

Temperature.— The  optimum  is  about  35°  C.  Pig- 
ment absent  in  cultures  that  develop  in  the  incubator. 

Behavior  to  Gelatin.— Liquefies  very  rapidly. 

Pigment  production.— Varies  greatly.  May  be 
grayish  to  bright  brick  red.  Usually  is  light  pink,  so 
that  present  cultures  may  be  considered  to  be  attenuated. 

Pathogenesis. — Has  marked  toxic  action,  and  when 
injected  in  large  amounts  into  the  abdominal  cavity,  or 
into  the  veins  of  rabbits  and  guinea-pigs,  proves  fatal. 
Rabbits  develop  marked  diarrhoea  and  die  in  from  3  to  20 
hours.  On  post-mortem  the  intestines  show  a  severe 
inflammatory  condition  of  the  mucous  membrane  and  at 
times  ulcerations. 


MEMORANDA. 


—  38  — 


BACILLUS  RUBER  OF  KIEL. 

J.    BREUNIG. — BACTERIOLOGISCHE     UNTERSUCHUXG     DES    TRIXKVVAS- 
SERS  DER  STADT    KIEL    (iNAUG. -DISSERTATION)   KIEL,  1888. 

EM.  LAURENT.— ANNALES  DE  L'INSTITUT  PASTEUR  IV,  464,  1890. 

Origin. — Drinking  waler  of  Kiel. 

Form. — Rods  about  three  to  five  or  seven  times  as 
long  as  wide. 

Motility. — Somewhat  motile,  and  the  motion  de- 
pends on  presence  of  oxygen. 

Sporulation.--Not  observed. 

Anilin  Dyes.— Stain  readily. 

Growth. — Rapid  and  abundant. 

Gelatin  Plates.— Deep  colonies  are  oval,  pale  yellow,  with  wavy  or  even 
border.  The  surface  colonies  are  blood  red  in  color,  spread  rapidly  and  have  a 
sinuous  border;  are  surrounded  by  a  clear  zone  and  liquefy  gelatin. 

Stich  Cultures. — Develop  along  the  line  of  inoculation  and  liquefaction 
takes  place.  The  fluid  becomes  strongly  colored  and  gas  may  form  in  the 
deeper  layers. 

Streak  Cultures. — On  agar,  at  30-35°,  the  growth  is  at  first  a  pale  rose,  and 
later  becomes  a  brick  red.  On  potatoes,  at  30-35°,  develop  rapidly,  forming  a 
purple  red  growth.  At  lower  temperatures  the  color  is  less  intense,  and  at 
first  is  orange,  later  carmine  red. 

Milk.— At  35°,  coagulation  takes  place  in  24  honrs,  without  a  trace  of 
coloration,  due  to  rapid  growth  and  production  of  acidity.  At  ordinary  tem- 
perature the  coagulation  takes  place  slowly  and  the  fluid  gradually  colors. 

Oxygen  requirements. — Is  a  facultative  anaerobe, 
but  requires  oxygen  to  form  the  pigment 

Temperature.— Grows  from  10  to  42°C.  The  opti- 
mum is  30-35°,  and  above  this  the  growth  ceases  to  be 
colored.  Direct  insolation  kills  it  in  5  hours.  By  exposure 
of  3  hours  is  not  killed  but  no  longer  produces  the  pig- 
ment, i.  e  ,  becomes  attenuated. 

Behavior  to  Gelatin.  —  Liquefies  gelatin  quite 
rapidly. 

Aerogenesis.— Some  gas  bubbles  may  form  in  gela- 
tin tubes. 

Pathogenesis. — No  action  observed. 


MEMORANDA, 


—  40  — 


BACILLUS  RUBIDUS. 

RED    BACILLUS    OF    WATER. 

Origin.— Water. 

Form. — Long-,  narrow  rod;  forms  threads. 
Motility.— Ac-lively  motile. 
Sporulation.— No  spores  observed. 
Anilin  Dyes.— Stain  readily. 
Growth. — Fairly  rapid. 

Gelatin  Plates.— Small,  yellow,  finely  granular  colonies,  with  irregular 
border.  Liquefies  gelatin. 

Stick  Cultures.— Liquefies  gelatin  slowly  along  line  of  inoculation.  The 
mass  of  bacteria  settles  to  the  bottom,  colored  yellowish  brown,  and  a  thin 
folded  scum  forms  on  the  surface. 

Streak  Cultures.— On  ogar,  thin,  irregular  bordered,  slightly  folded  and 
colored  growth.  On  potatoes,  the  most  characteristic  growth  is  developed, 
which  spreads  and  lias  a  bright  brick  red  color.  On  blood  serum,  liquefaction 
takes  place  and  red  pigment  forms. 

Oxygen  requirements.— Is  aerobic. 

Temperature. — Does  not  grow  at  the  temperature 
of  body. 


Behavior  to  Gelatin.— Liquefies. 
Pathogenesis. — No  effect  observed. 


MEMORANDA. 


42  — 


BACILLUS  VIOLACEUS. 

VIOLET  BACILLUS  OF  AVATEIJ. 

Origin. — Water  of  the  river  Spree  at  Berlin,  and  of 
the  Thames  at  London  ;  also  in  well  water. 

Form. — Long,  narrow  rods,  about  three  times  as  long 
as  wide ;  forms  threads. 

Motility. — Actively  motile. 

Sporulation. — Forms  median  spores. 
Anilin  Dyes.— React  readily. 
Growth. — Is  moderately  rapid. 

Gelatin  Plates.— Irregular  colonies,  with  loose  fibrillaled  borders.  The 
center  shows  quite  early  a  violet  color.  Liquefies. 

Stich  Cultures.—  Funnel-shaped  liquefaction  along  entire  line  of  inocu- 
lation. A  violet  sediment  collects  on  the  bottom,  while  the  liquefied  gekxtin 
above  is  perfectly  clear. 

Streak  Cultures. — On  agar,  forms  a  thin,  moist,  bright  violet  covering. 
On  potatoes,  the  growth  is  somewhat  slow  but  very  characteristic,  forming  a 
bright  violet,  eventually  dark  covering  On  blood  serum,  the  violet  color  is 
produced  and  liquefaction  takes  place. 

Oxygen  requirements. — Is  a  facultative  anaerobe. 
Oxygen  is  necessary  to  pigment  formation. 

Temperature.  —  Does  not  grow  at  higher  tem- 
peratures. 

Behavior  to  Gelatin. — Liquefies. 
Pathogenesis.— Has  no  effect. 


MEMORANDA. 


—  44  — 


BACILLUS  FLUORESCENS  PUTIDTJS.    Fliigge. 

FLUORESCING    BACILLUS    OF    WATER. 

Origin.— Putrid  media,  water. 

Form.— Short,  small  rods,  with  rounded  ends. 

Motility. — Very  actively  motile. 

Speculation.—  No  spores  observed. 

Anilin  Dyes.— Stain  readily. 

Growth.— Rapid . 

Gelatin  Plates.— Deep  colonies  are  small,  round,  finely  granular.  Surface 
colonies  spread  rapidly  and  form  at  first  a  very  thin  plaque,  with  irregular, 
wavy  border,  which  shows  markings.  Later  a  bluish  green  color  diffuses 
through  the  surrounding  gelatin.  Odor  of  trimethylamine.  No  liquefaction. 

Stich  Cultures.— No  growth  in  lower  part  of  tube.  Surface  of  gelatin, 
covered  with  grayish  white  growth,  while  the  fluorescing  pigment  gradually 
diffuses  downward  into  the  gelatin. 

Streak  Cultures.— On  agar,  a  moist,  spreading  growth.  The  agar  becomes 
colored,  but  later  the  color  fades.  On  potatoes,  a  thin  grayish  or  brownish, 
moist  growth  lorms. 

Oxygen  requirements.— Aerobic. 
Temperature. — Ordinary  room  temperature  is  best. 
Behavior  to  Gelatin.— Does  not  liquefy. 
Pathogenesis.— Without  action. 


MEMORANDA, 


—  46  — 


BACTERIUM  PHOSPHORESCENS.     Fischer. 

THIS    IS  BUT  ONE  OF    A    NUMBER  OF    BACTERIA  FOUND    IN    SEA-WATER 

WHICH  POSSESSES  THE  PROPERTY  OF  PHOSPHORESCING  IN  THE  DARK. 
PHOTOBACTEBIUM. 

Origin.— In  water  of  the  harbor  of  Kiel,  also  on  sea 
fish. 

Form. — Short,  thick  bacillus,  with  rounded  ends; 
sometimes  almost  a  coccus.  Usually  in  pairs,  may  form 
threads.  Involution  forms  soon  develop. 

Motility. — No  motion. 

Sporulation.— Not  observed. 

Anilin  Dyes.— Slain  readily. 

Growth. — Moderately  rapid,  and  the  cultures  show 
a  greenish  phosphorescence  in  the  dark. 

Gelatin  Plates.— Show  small,  white,  glistening  colonies,  which  do  not 
liquefy  gelatin.  The  border  is  sharp,  irregular,  and  contents  are  granular, 
and  show  several  concentric  rings. 

Stich  Cultures.- -Granular  growth  along  the  line  of  inoculation,  but  is 
most  abundant  on  the  surface,  forming  a  thin  grayish  white  covering.  Even- 
tually the  gelatin  is  colored  a  yellowish  brown. 

Streak  Cultures. — On  agar,  potatoes,  etc.,  growth  is  limited  to  the  line  of 
inoculation.  Grows  also  well  on  fish,  beef,  bread,  fats,  etc. 

Oxygen  requirements.— Is  a  facultative  anaerobe. 
The  production  of  light  depends  upon  the  presence  of 
oxygen,  and  is  therefore  most  marked  on  the  surface 
growths.  The  intensity  of  the  iight  may  diminish  and 
eventually  become  lost — attenuation.  May  be  restored 
by  growth  on  suitable  media,  as  salt  fish,  etc. 

Temperature. — Does  not  grow  in  incubator. 
grow  at  0°  C. 

Behavior  to  Gelatin. — Does  not  liquefy. 
Pathogenesis. — No  effect  on  animals. 


MEMORANDA. 


—  48  — 


SARCINA  AURANTIACA. 

ORANGE    SAKCIXE. 

Origin. — From  air,  weiss-beer. 

Form. — Small,  spherical  cocoi,  grouped  in  2  and  4, 
and  also  forming  package-shaped  masses. 

Motility. — None. 

Sporulation. — None. 

Anilin  Dyes. — Slain  very  easily  and  are  likely  to 
over  stain. 

Growth.— Rather  rapid. 

Gelatin  Plates.— Show  round,  sharp-edged  colonies,  which  are  granular 
and  of  an  orange-yellow  color.  Liquefy. 

Rtich  Cultures.— Liquefies  gelatin  along  entire  line  of  inoculation.  Even- 
tually an  orange-colored  deposit  of  bacteria  forms  on  the  bottom  and  the 
liquid  above  becomes  clear. 

Streak  Culture^ — ')n  (if/or,  forms  a  thick,  orange-colored  growth.  On 
potatoes,  the  pigment  i.s  excellently  developed. 

Oxygen  requirements.— Is  aerobic. 
Temperature. — Higher  temperatures  unfavorable. 
Behavior  to  Gelatin.— Liquefies  rapidly. 
Aerogenesis. — Not  observed. 
Pathogenesis.— Has  no  effect. 


MEMORANDA. 


—  50  — 


SARCINA  LUTEA.     Schroter. 

YELLOW    SARCIXE. 

Origin. — Air. 

Form. — Larger  cocci  than  the  orange  sarcine  and 
forms  more  perfect  package-shaped  masses. 

Motility.— None. 
Sporulation.— None. 

Anilin  Dyes. —React  readily  and  are  likely  to  over- 
stain. 

Growth. — Very  slow. 

Gelatin  Plates.— Colonies  develop  very  slowly  as  minute  yellowish  spots, 
which  show  an  irregular  outline  and  are  markedly  granular.  The  colonies  do 
not  liquefy  gelatin. 

SticJt  Cultures. — Gi'owth  is  especially  developed  on  the  surface  and  ex- 
tends but  slightly  down  the  line  of  inoculation.  Lower  half  of  tube  is  usually 
free  from  growth.  The  color  is  bright  yellow  and  in  very  old  tubes  liquefaction 
slowly  shows  itself,  so  that  eventually  a  bright  yellow  deposit  forms  on  the 
bottom  while  the  liquefied  gelatin  above  is  perfectly  cjear. 

Streak  Cultures. — On  a<t«r,  forms  a  very  thick,  moist,  bright  yellow  cov- 
ering. On  potatoes,  the  growth  is  slow,  with  production  of  same  color. 

Oxygen  requirements. — Aerobe. 
Temperature. — May  grow  in  the  incubator. 

Behavior  to  Gelatin.— Very  slow  liquefaction,  ap- 
pearing after  lapse  of  a  week  or  two. 

Pathogenesis. — None. 


MEMORANDA. 


BACILLUS  SUBTILIS.     Ehrenberg. 

HAY    BACILLUS. 

Origin. — In  air,  water,  soil,  feces,  putrid  fluids,  and 
in  infusions  of  hay. 

Form. — Lame,  rather  narrow  rods,  about  3  times  as 
long  as  wide,  and  with  rounded  ends.  Forms  threads. 

Motility. — Actively  motile.  Has  a  flagellum  at 
each  end. 

Sporulation. — Forms  large,  oval  spores  at  or  near 
the  middle,  without  enlargement.  Are  highly  resistant 
and  can  be  readily  double  stained. 

Anilin  Dyes. — Stain  readily. 

Growth. — Very  rapid.  At  21°  0.  the  division  of  a 
cell  has  been  observed  to  take  place  in  1£  hours,  and  at 
35°  in  20  minutes. 

Gelatin  Plates.— The  surface  colonies  liquefy  gelatin  rapidly  and  exten- 
sively and  present  a  characteristic  appearance.  The  central  portion  appears 
as  a  grayish  yellow,  irregular  mass,  which  enclose  examination  can  be  seen 
to  be  made  up  of  moving  cells.  The  border  of  the  colony  is  quite  character- 
istic. It  consists  of  a  dense  zone  of  bacilli  and  threads,  radially  arranged,  so 
that  the  ends  project  outward,  thus  presenting  a  peculiar  appearance— the  so- 
called  "ray  crown."  . 

Stick  Cultures.— Very  rapid  liquefaction  takes  place  along  the  entire  line 
of  inoculation.  White  flocculent  masses  accumulate  at  the  bottom  while  the 
liquid  above,  at  first  turbid,  becomes  clear.  On  the  surface  a  dense  white 
scum  or  zoogloea  usually  forms. 

Streak  Cultures. — On  ayur,  forms  a  grayish  white,  thick,  folded  scum. 
On  potatoes,  develops  excellently,  and  forms  a  moist,  thick,  yellowish-white 
covering,  which  at  first  is  velvety  in  appearance  but  later  becomes  dry  and 
granular,  and  contains  spores  as  well  as  involution  forms.  On  blood  serum, 
forms  also  a  folded  scum  and  liquefies. 

Oxygen  requirements. — Is  aerobic. 

Temperature. — Grows  from  10  to  45°.  Optimum 
about  30°C. 

Behavior  to  Gelatin. — Liquefies  rapidly  and  exten- 
sively. 

Pathogenesis. — Has  no  pathogenic  power.  Large 
numbers  of  spores  injected  into  the  blood  soon  disappear 
and  are  taken  up  by  the  liver  and  spleen.  They  may  be 
stored  up  in  these  organs  for  60  to  70  days  and  yet  pre- 
serve their  vitality  (Wyssokowitsch). 


MEMORANDA, 


—  54  — 


BACILLUS  MESENTEBJCUS  VULGATUS. 

Flugge. 

POTATO  BACILLI'*.       A    GKOl'P    OF  ' 'POTATO  BACILLl"   ARE  KNOWN  WHICH 
ll.\\'E  MANY  CIIAIIACTKRISTIC.S  IN  COMMON. 

Origin. — Widely  distributed  in  the  soil,  on  surface 
of  potatoes  in  feces,  putrid  fluids,  water,  milk. 

Form. — Small,  thick  rods,  with  rounded  ends,  usually 
in  pairs,  inny  form  threads. 

Motility. — Actively  motile. 

Sporulation. — Readily  forms  median,  round  spores. 
The  spores  of  one  variety  of  "  potato  bacillus"  described 
by  Globig.  shoved  enormous  powers  of  existence,  with- 
standing the  action  of  steam-heat  for  5  to  6  hours. 

Anilin  Dyes.— React  easily. 

Growth.— Rapid. 

(,'i'Ut/in  7'Iott'x. — Show  yellowish  white,  slightly  granular  colonies,  with 
irregular  borders.  Liquefy  rapidly  and  extensively. 

,S7/'>/<  Cultures.— Growth  occurs  along  the  entire  line  of  inoculation,  but 
liquefaction  is  more  energetic  in  the  upper  part.  The  liquefied  gelatin 
remains  turbi'd  for  some  time  and  a  thin,  grayish,  folded  scum  forms  on  the 
top. 

.S7/v«/r  CtiUii'rt'N.—On  ac/(ir,  forms  a  dull  white  or  grayish,  folded  growth. 
On  potato!*,  the  most  characteristic  growth  develops.  The  surface  is  rapidly 
covered  with  a  thick,  white,  strongly  folded,  coherent  growth.  Later  the 
color  become  s  ;i  dirty  brown  or  red. 

Milk. — 1«  coagulated. 

Oxygen  requirements. — A e robe. 

Temperature. — ({rows  at  ordinary  as  well  as  higher 
temperatures. 

Behavior  to  Gelatin.— Liquefies  rapidly. 
Pathogenesis. — No  effect  observed. 


MEMORANDA. 


—  56  — 


BACILLUS  MEGATERITJM.    De  Bary. 

Origin. — From  boiled  cabbage  leaves. 

Form. — Cylindrical  rods,  with  granular  contents,  3 
to  6  times  as  long  as  broad,  with  rounded  ends.  Are 
usually  slightly  bent  and  may  form  threads.  Involution 
forms  common. 

Motility. — Slow,  amseboid  motion.     Lateral  flagella. 
Sporulation. — Forms  median  spores. 
Anilin  Dyes. — Stain  readily,   though  irregularities 
due  to  granular  protoplasm  may  be  seen. 
Growth. — Rapid. 

Gelatin  Plates.— Colonies  are  at  first  irregular,  small,  yellowish  masses, 
but  subsequently  show  marked  radiating  or  branching  forms,  which  soon 
Mqnef  y  the  gelatin . 

Stick  Cu Itures.— Rapid  growth  and  liquefaction  along  the  line  of  inocu- 
iution.  May  show  threads  of  bacteria  penetrating  outward  into  the  solid  gel- 
atin. Eventually  the  gelatin  is  wholly  liquefied  and  a  flocculent  mass  accu- 
mulates on  the  bottom;  the  supernatant  liquid  clears  up  without  formation 
of  scum  on  top. 

Streak  Cultures.— On  agar,  forms  a  dull  white  or  grayish  covering.  On 
potatoes,  grows  rapidly  as  a  thick,  slimy,  grayish  white  mass,  which  is  rich  in 
spores  and  involution  forms. 

Oxygen  requirements. — Is  aerobic. 
Temperature.— Optimum    about  20°C.    May  grow 
in  incubator. 

Behavior  to  Gelatin.— Liquefies. 
Pathogenesis.— No  effect  observed. 


MEMORANDA. 


—  58  — 


BACILLUS  RAMOSUS. 

ROOT    OR    WURZEL  BACILLUS. 

Origin. — Very  common  in  earth  ;  occurs  also  in  river 
and  spring  water. 

Form. — Rather  large  rod?,  thicker  than  the  Hay 
bacillus;  with  slightly  rounded  ends.  Threads  common. 

Motility.— Slowly  motile. 
Sporulation. — Large  median  spores  occur. 
Anilin  Dyes.— Stains  well. 
Growth.  —Rapid. 

Gelatin  Plates.— The  colonies  present  a  characteristic  appearance,  resem- 
bling somewhat  fine  branching  rootlets,  hence  the  name.  At  first  the  colo- 
nies are  round,  dark  and  with  bristly  borders.  Subsequently  the  colonies 
branch  and  ramify  throughout  the  gelatin  which  is  liquefied. 

Stick  Cultures.— Are  also  characteristic.  Growth  develops  along  the  line 
of  inoculation  and  from  this  threads  penetrate  or  radiate  into  the  surround- 
ing gelatin.  The  growth  is  more  rapid  at  the  top  than  in  the  lower  parts  of 
the  tube  so  that  the  appearance  of  an  "inverted  pine  tree"  results.  Later 
the  gelatin  is  liquefied  completely.  The  bacterial  growth  accumulates  on  the 
bottom  while  the  Ijquid  above  becomes  clear  and  has  a  thin  scum  on  the 
surface. 

Streak  Cultures.— On  agar,  forms  a  grayish  growth,  spreading  outward 
from  the  streak  so  that  the  appearance  often  is  not  unlike  thatof  a  centipede. 
On  potatoes,  a  slimy,  whitish  growth  which  develop  spores. 

Oxygen  requirements.— Is  aerobic. 

Temperature. — Grows  at  ordinary  temperature  and 
also  in  incubator. 

Behavior  to  Gelatin.— Liquefies. 

Pathogenesis. — Without  effect,  even  in  very  large 
doses. 


MEMORANDA. 


—  60  — 


PROTEUS  VULGARIS.    Hauser. 

INCLUDED  IN  THE  BACTERIUM  TERMO  OF  OLDER  WRITERS. 

Origin. — Very  widely  distributed.  Is  commonly 
present  in  the  putrefaction  of  animal  proteids;  has  also 
been  met  with  in  water,  in  meconium,  in  purulent  ab- 
scesses, and  in  blood  and  tissues  of  two  cases  of  fatal 
putrid  infection  of  intestines. 

Form. — Rods,  of  varying  length,  from  short  oval 
forms  to  those  which  are  2  to  6  times  as  long  as  wide.  It 
is  usually  bent  and  grows  in  pairs;  may  also  form  twisted, 
interwoven  threads.  Roundish  involution  forms  are 
common. 

Motility. — Actively  motile.  Flagella  very  numerous 
and  lateral. 

Sporulation. — Not  observed,  though  cultures  are  re- 
sistant 1o  desiccation  and  retain  vitality  for  many  months. 

Anilin  Dyes. — Stain  readily. 

Growth. — Very  rapid. 

Gelatin  Plates.— Rapid  and  extensive  liquefaction  of  the  gelatin.  The 
colonies  are  yellowish  brown,  with  bristly  borders,  and  in  soft  gelatin  tend  to 
spread  over  the  surface  and  assume  peculiar  figures.  Detached  portions  of 
colonies  can  be  seen  to  move  about— "swarming  islets."  Disagreeable  odor 
and  alkaline  reaction. 

Stich  Cultures. — Rapid  liquefaction  along  entire  line  of  inoculation,  so 
that  in  a  few  days  the  entire  contents  are  liquefied.  The  fluid  is  at  first  dif- 
fusely cloudy,  but  later  clears  up  and  a  flocculent  sediment  settles  on  the  bot- 
tom, while  on  ihe  top  a  grayish  white  layer  is  formed. 

Streak  Cultures.— On  agar,  forms  a  grayish,  slimy,  rapidly  spreading 
growth.  On  potatoes,  it  forms  a  dirty  colored,  sticky  covering. 

Oxygen  requirements.— Facultative  anaerobe. 

Temperature. — Optimum  lies  between  20  and  24°. 
Grows  excellently  in  the  incubator. 

Behavior  to  Gelatin. — Rapidly  liquefied. 

Aerogenesis. — Forms  hydrogen  sulphide. 

Pathogenesis. — Small  doses  have  no  effect.  Injec- 
tion of  large  quantities  of  cultures,  or  filtrates  from  these, 
produces  in  rabbits  and  guinea-pigs  toxic  effects,  and  even 
death  may  result.  It  is  therefore  toxicogenic,  but  not 
pathogenic. 

NOTE.— When  surface  colonies  as  those  above  present  special  character- 
istic they  can  be  reprinted  on  cover-glasses.  To  make  such  an  impression  or 
"Klatsch"  preparation,  select  a  suitable  spreading  colony,  wilh  the  aid  of  No 
3  objective,  then  raise  the  lube  of  the  microscope  and  carefully  drop  a  clean- 
cover-glass  on  top  of  the  colony.  Apply  gentle  pressure  with  a  pair  of  forceps, 
then  grasp  the  edge  of  the  cover-glass  and  carefully  remove:  allow  to  dry  in 
the  air;  fix  and  stain  in  the  usual  manner. 

In  making  the  reprint  only  the  growth  should  adhere  to  the  cover-glass. 
Considerable  gelatin,  solid  or  liquid,  on  the  cover-glass,  is  undesirable  and 
interferes. 


MEMORANDA. 


—  62  — 


BACTERIUM  ZOPFII.     Kurth. 

Origin. — From  the  intestines  of  chicken. 

Form. — Rods,  2  to  5  times  as  long  as  wide.  Forms 
threads,  which  in  gelatin  are  often  peculiarly  bent  or 
twisted. 

Motility. — Actively  motile. 

Sporulation. — Spore-like  bodies  are  formed,  which 
resist  desiccation,  but  are  readily  destroyed  by  heat,  and 
are  readily  stained  by  anilin  dyes. 

Anilin  Dyes. — Stain  easily. 

Growth.— Rapid. 

Gelatin  Plates.— The  colonies  form  delicate  cloudy  patches  of  radiating 
threads,  and  under  the  microscope  show,  in  addition  to  the  network  of 
threads,  numerous  rounded  little  masses  or  bunches  of  cells. 

Stick  Cultures.— Marked  growth  in  the  upper  part  of  the  tube  and  almost 
absent  in  the  lower  part.  Shows  fine  radiating  lines  which,  at  or  near  the 
surface,  penetrate  deepest  into  the  surrounding  gelatin. 

Streak  Cultures.— On  agar,  forms  a  very  thin,  dry,  grayish  growth. 

Oxygen  requirements. — Is  aerobic. 

Temperature. — Grows  best  at  ordinary  temperature. 
Can  grow  at  37-40°,  but  tends  to  develop  involution  forms 
and  to  die  out. 

Behavior  to  Gelatin. — Is  not  liquefied. 

Pathogenesis. — No  effect  on  animals. 

NOTE.— Make  "Klatsch"  or  impression  preparations  of  the  colonies. 


MEMORANDA. 


—  64  — 


SPIRILLUM  RTJBRTJM.    Von  Esmarch. 

Origin. — Isolated  from  the  putrefied  cadaver  of  a 
mouse. 

Form. — Clear,  transparent,  thick  cells,  which  com- 
monly are  single,  appearing  as  large  bent  rods  or  comma 
bacilli  (vibrio).  May  form  spirals  of  3  or  4  or  even  40 
windings.  Involution  forms  are  common  in  old  cultures. 

Motility.  —Actively  motile.  Each  end  of  a  spiral 
has  one  wavy  flagellum. 

Sporulation. — True  spores  not  observed. 
Anilin  Dyes.— Stain  slowly  but  well. 
Growth.— Extremely  s?o\v. 

Gelatin  Plate 8  — Owing  to  the  very  slow  development  of  colonies  ordinary 
plates  cannot  be  used.  In  roll  tubes,  colonies  develop  in  from  7  to  10  days,  and 
at  first  are  minute  and  grayish;  later  the  center  becomes  tinged  with  pink 
and  eventually  becomes  red.  The  edge  is  smooth  and  contents  finely 
granular. 

Stick  Cu//?nvx.— Are  the  most  characteristic.  Growth  takes  place  along 
the  entire  line  of  inocukvtion,  forming  a  row  of  colonies.  The  growth  spreads 
slightly  on  the  surface  and  is  colored  a  light  pink.  The  pigment  formation  is 
most  marked  along  the  stich— where  oxygen  is  absent.  It  passes  through  a 
light  pink  to  a  beautiful  dark  wine-red  color.  Ordinary  bacterial  pigments 
are  formed  only  in  the  presence  of  air  and  are  secondary  products,  whereas 
this  pigment  is  formed  in  the  absence  of  air  and  is  primary. 

Streak  Cultures.— On  agar,  forms  moist,  thick,  non-spreading  patches, 
which,  when  old,  possess  a  light  pink  or  red  color,  especially  near  the  center. 
On  potatoes,  develops  slowly,  forming  minute  deep  red  colonies.  On  blood 
serum,  the  growth  is  much  the  same  as  on  agar. 

Milk. — In  fluid.media,  milk,  bouillon,  etc.,  forms  long  spirals. 

Oxygen  requirements.— Is  a  facultative  anaerobe. 

Temperature.— Grows  between  16°  and  40°.  Opti- 
mum about  37°0. 

Behavior  to  Gelatin. — Not  liquefied. 
Pathogenesis.— Has  no  effect. 

NOTE.— Make  Esmarch  Roll-tubes  of  the  Spirillum  rubrum. 


MEMORANDA, 


—  66— 


BACILLUS  ACIDI  LACTICI.     Hueppe. 

BACILLUS  OF    LACTIC    ACID    FERMENTATION.       IS    ONLY    ONE    OF    A   LARGE 
NUMBER  OF  BACTERIA  GIVING  RISE  TO  LACTIC  ACID. 

Origin.— Sour  milk. 

Form. — Short,  thick  rods,  about  one-half  as  long  as 
long  as  wide;  usually  in  pairs,  rarely  in  chains. 

Motility. — Has  no  motion.  Brownian  movement, 
however,  is  marked. 

Sporulation. — Hound,  terminal  spores  observed. 

Anilin  Dyes. — Stain  readily. 

Growth.— Abundant  and  fairly  rapid. 

Gelatin  Plates.— The  deep  colonies  are  round  or  oval,  yellow,  sharp  bor- 
dered, finely  granular.  The  surface  colonies  spread,  forming  thin  plaques, 
with  irregular,  wavy  borders.  The  outer  zone  of  the  colony  is  at  first  almost 
transparent  and  shows  markings  resembling  the  venation  of  leaves. 

Stich  Cultures. — Slight  growth  along  the  stich,  but  on  the  surface  it  is 
considerable  and  spreads  rapMly  as  in  thin,  dry,  pearly-white  covering.  In 
old  cultures  bundles  of  crystals  form  along  the  stich  at  or  near  the  surface. 

Streak  Cultures.— On  agar,  forms  a  grayish  white,  moist,  spreading 
growth,  which  offers  no.  special  characteristics.  On  potatoes,  it  forms  a 
brownish  yellow,  slimy  covering. 

31Wc. — In  sterilized  milk  converts  the  lactose  or  milk-sugar  into  lactic 
acid  and  carbonic  acid.  The  acid  reaction  thus  produced  causes  a  precipita- 
tion of  the  casein  or  curd.  This  change  occurs  only  in  presence  of  air. 

Oxygen  requirements. — Is  a  facultative  anaerobe. 
Temperature.— Grows  between  10°  and  45°.    Opti- 
mum about  35°0. 

Behavior  to  Gelatin.— Not  liquefied. 

Aerogenesis. — Gas  is  produced  in  milk. 

Pathogenesis.— No  effect.  0.75  per  cent,  lactic  acid 
stops  the  growth.  Production  of  lactic  acid  in  the  mouth 
and  dental  caries ;  abnormal  fermentations  in  the  stomach, 
in  the  intestines.  Lactic  acid  bacteria  favor  the  growth 
of  anaerobic  bacteria. 


MEMORANDA. 


—  68— 


BACILLUS  BUTYRICUS.    Hueppe. 

BACILLUS  OF  BUTYRIC  ACID  FERMENTATION.      IS  ONLY    ONE    OF    A   LARGE 
NUMBER    OF    AEROBIC  AND  ANAEROBIC  BACTERIA    WHICH    GIVE  RISE 
TO  BUTYRIC  ACID.      THE  VIBRION    BUTYRIQUE    OF    PASTEUR 
WAS  THE  FIRST  ANAEROBE  DISCOVERED  (1861). 

Origin.— Milk. 

Form. — Lon  •;,  narrow  rods,  with  rounded  ends ,  fre- 
quently in  pairs,  may  form  threads. 

Motility. — Actively  motile. 

Sporulation.— At  about  30°  forms  bright,  oval,  me- 
dian spores. 

Anilin  Dyes.— React  well. 

Growth. — Rapid. 

Gelatin  Plates.— The  deep  colonies  form  yellowish  masses,  whereas  the 
surface  ones  liquefy  rapidly  and  then  form  grayish-brown,  granular  patches 
with  fibri Hated  borders. 

Mich  Cultures.— Rapid  liquefaction  along  entire  line  of  inoculation.  The 
gelatin  becomes  colored  yellowish  and  on  the  surface  a  thin,  folded,  grayish 
white  scum  forms.  The  liquid  remains  cloudy  for  some  time  but  later  the 
growth  settles  to  the  bottom. 

Streak  Cultures.— On  agar,  forms  a  light,  yellow,  sticky  covering.  On 
potatoes,  forms  a  light  brown,  transparent  growth  which  sometimes  becomes 
folded. 

Milk.— Without  change  in  the  amphoteric  reaction  the  casein  gradually 
coagulates,  as  with  rennet.  Subsequently  after  about  8  days  the  casein  is 
redissolved  or  peptonized  with  formation  of  pepton,  leucin,tyrosin,  ammonia 
and  bitter  products.  From  hydrated  milk  sugar  and  lactates  it  forms  butyric 
acid. 

Oxygen  requirements. — Is  aerobic. 
Temperature. — Can  grow  at  ordinary  temperature, 
but  its  optimum  is  35  to  40°  C. 

Behavior  to  Gelatin.— Liquefies. 
Aerogenesis. — Butyric  acid  formed. 
Fathogenesis. — No  effect. 


MEMOEANDA, 


—  70  — 


BACILLUS  CYANOGENUS.     Fuchs,  (1841). 

BACILLUS  OF  BLUE  MILK. 

Origin.— In  blue  milk. 

Form. — Small,  rather  narrow  rod.5,  with  slightly 
rounded  ends,  2  to  3  times  as  long  as  wide.  Frequently 
grows  in  pairs,  very  rarely  in  threads. 

Motility. — Very  actively  rnolile. 

Sporulation. — Small  terminal  spores  observed  in 
gelatin,  milk,  etc.,  at  ordinary  temperature. 

Anilin  Dyes.— Stain  easily. 

Growth. — Rapid. 

Gelatin  Plates, — The  deep  colonies  are  round  with  sharp,  smooth  border, 
and  yellowish  granular  contents.  The  surface  colonies  are  moist,  elevated, 
convex  masses,  which  are  round,  finely  granular  and  dark  colored. 

Stich  Cultures  — Little  or  no  growth  in  the  lower  part  of  the  stich. 
Spreads  over  the  surface  as  a  thick,  moist,  dark  gray  covering.  A  dark  steel- 
blue  color  diffuses  downward  into  the  gelatin.  The  shade  of  color  varies  with 
the  reaction  of  the  medium.  In  neutral  or  acid  media  it  is  quite  blue,  whereas 
in  very  alkaline  media  it  is  dark  or  even  black.  The  cultures  when  old 
become  dark  colored. 

Streak  Cultures.— On  agar,  forms  a  dirty  gray,  thick,  moist  covering, 
and  the  medium  becomes  diffusely  colored.  On  potatoes,  it  likewise  forms  a 
thick,  raised,  slimy  growth,  which  rapidly  spreads  and  becomes  colored.  On 
blood  scrum,  no  color  is  formed. 

Milk. — In  sterilized  milk  it  produces  no  acid  or  coagulation,  but  the 
liquid  becomes  colored  a  slate  gray  which  with  acids  turns  blue,  In  unsteril- 
ized  milk,  that  is  in  presence  of  lactic  acid  bacteria,  the  color  is  sky-blue. 
The  color  is  developed  from  casein,  not  from  lactose. 

Oxygen  requirements.— Aerobic. 

Temperature. — Can  grow  at  ordinary  temperature, 
or  in  incubator.  The  pigment  is  best  developed  at  low 
temperatures,  15-18°C. 

Behavior  to  Gelatin. — Not  liquefied. 

Pathogenesis.— No  effect  on  animals. 

NOTE.— Make  "  Kiatsch  "  or  impression  preparations  of  the  colonies. 


MEMORANDA. 


—  72  — 


OIDIUM  LACTIS. 

DOES  NOT  BELONG  TO  THE  BACTERIA,  BUT  IS  A  SIMPLE  MOULD. 

Origin. — Almost  invariably  present  in  milk  and  in 
butler. 

Form. — A  delicate  white  mycelium  of  wavy  threads. 
No  special  fruit  organ.  Large  oblong  spores. 

Anilin  Dyes. — Eeact  readily. 
Growth. — Rapid. 

Gelatin  Plates. — Delicate  white  stars  form,  which  rapidly  enlarge,  and  on 
the  surface  spread  as  flat,  whitish,  dry  masses.  Under  the  microscope  the 
colonies  show  radiating  branched  hyphse. 

Stich  Cultures.— Growth  takes  place  along  the  entire  line  of  inoculation, 
but  most  abundantly  at  or  near  the  surface.  A  branching  network  of  threads 
extends  outward  into  the  solid  gelatin.  On  the  surface  a  grayish  white,  dry, 
low  growth  forms.  In  old  cultures  only  the  upper  layer  of  gelatin  shows  the 
radiating  lines. 

Streak  Cultures.— On  agar,  it  forms  a  grayish  white,  thin  growth. 

Milk.— Growth  occurs  without  any  change  in  its  composition. 

Temperature. — Grows  best  at  ordinary  temperature. 
Can  grow  in  inc.ubator. 

Behavior  to  Gelatin. — Does  not  liquefy. 
Pathogenesis. — No  effect  on  animals. 


MEMORANDA. 


MEMORANDA. 


—  73  — 


BACTERIOLOGICAL,    EXAMINATION    OF 
WATER. 

The  water  to  be  examined  must  be  received  in  a 
sterilized  bottle  or  flask,  thoroughly  protected  against 
subsequent  contamination.  Furthermore,  in  view  of  the 
rapid  multiplication  of  bacteria,  a  given  sample  of  water 
should  be  examined  as  soon  as  possible  after  collection. 
The  method  commonly  employed  consists  in  the  deter- 
mination of  the  number  of  bacteria  present  in  a  given 
volume,  1  c.  c.,  and  the  recognition  of  the  several  species  or 
kinds  of  microorganisms  present.  This  process,  as  carried 
out,  is  as  follows : 

Place  several  1  c.  c.  pipettes,  graduated  in  1-10  c.  c., 
in  a  pipette  box  and  sterilize  in  the  dry  heat  oven  in  the 
usual  way.  Liquefy  3  gelatin  tubes  and  with  a  sterilized 
cooled  pipette  transfer  into  tube  No.  1  one  c.c.  of  the  water ; 
into  tube  No.  2  place  one-half  c.c.,  and  into  tube  No.  3 one 
drop  of  the  water.  Gently  agitate  the  contents  of  the 
tubes,  to  secure  complete  mixture,  then  pour  the  gelatin 
onto  sterilized  glass  plates,  observing  the  usual  precau- 
tions in  making  plate  cultures.  Set  aside  the  gelatin 
plates  thus  obtained  for  two  or  three  days  and  then  count 
the  colonies  when  sufficiently  developed. 

When  only  a  small  number  of  colonies  are  present 
the  counting  can  be  done  with  the  unaided  eye,  but  when, 
as  it  frequently  happens,  the  number  is  very  large,  it  is 
desirable  to  make  use  of  a  counting  apparatus — that  of 
Wolffhiigel  is  usually  employed.  The  gelatin  plate  on 
which  the  colonies  are  to  be  counted  is  placed  on  the 
black  glass  base  and  covered  with  a  glass  plate  ruled  into 
squares.  The  number  of  colonies  under  six  or  more 
squares  is  thus  easily  determined,  and  in  this  way  the 


—  74  — 

average  number  of  colonies  per  square  readily  ascertained. 
By  determining  the  number  of  squares  which  the  gelatin 
on  the  plate  covers,  and  multiplying  this  figure  by  the 
average  number  of  colonies  per  square,  the  total  number  of 
colonies  on  the  plate  is  found.  Since  each  colony  is  derived 
from  a  single  cell  this  number  then  represents  the  number 
of  bacteria  present  in  1  c.  c.  or  ^  c.  c.  or  1  drop  of  the 
water.  The  number  of  bacteria  found  should  always  be 
expressed  as  so  many  per  c.  c. 

To  ascertain  the  kind  of  bacteria  present,  the  colonies 
are  examined  under  the  microscope  in  the  usual  way.  A« 
seen  from  the  preceding  work  the  form  of  the  colony 
and  its  behavior  to  gelatin  may  sometimes  assist  in  its 
identification.  Hanging-drop  examinations,  stained  prepa- 
rations and  stich  cultures  will  still  further  assist  the 
recognition. 

The  chief  object  of  the  bacteriological  examination  of 
water  is  to  determine  the  presence  or  absence  of  patho- 
genic or  toxicogenic  bacteria.  In  the  above  method  this  is 
done  by  recognizing  the  colony  of  the  specific  organism 
sought  for.  When  the  pathogenic  bacteria,  as  the  cholera 
or  typhoid  fever  bacillus  for  example,  are  present  in  large 
numbers,  and  this  is  very  rarely  the  case,  the  identification 
can  perhaps  be  easily  d*one.  On  the  other  hand  a  few 
pathogenic  bacteria  in  the  presence  of  a  large  number  of 
saprophytic  organisms  can  be  easily  overlooked,  and  in 
such  cases  their  recognition  becomes  well-nigh  impossible. 
In  view  of  these  facts  the  following  method  has  been 
devised  and  used  in  this  laboratory  since  1888.  It  is  based 
upon  the  fact  that  the  majority  of  bacteria  present  in 
water  are  common  saprophytes  which  grow  at  the  ordi- 
nary temperature,  cannot  grow  at  the  temperature  of  the 
body,  and  cannot,  therefore,  produce  toxic  or  pathogenic 
effects.  Further,  that  those  bacteria  which  can  develop 
at  the  temperature  of  the  body  may  or  may  not  be  patho- 
genic, and  this  is  ascertained  by  animal  experiment.  The 
process  as  used  is  as  follows  : 


MEMORANDA. 


MEMORANDA. 


—  /o  — - 

To  sterilized  beef  tea  or  bouillon  tubes  add  1  c.  c.,  ^  c. 
c.,  and  one  drop  of  the  water  by  means  of  a  sterilized 
pipette.  Set  aside  in  the  incubator  at  37  to  39°  C.  for  24 
hours.  If  no  growth  occurs  at  this  temperature  it  is  at 
once  sufficient  evidence  that  the  water  is  free  from  disease- 
producing  organisms.  On  the  other  hand,  if  growth 
develops,  injections  of  1  c.  c.  of  the  culture  are  made  intra- 
peritoneally  into  white  rats  by-  means  of  a  sterilized  Koch 
syringe.  The  recovery  of  the  animal  indicates  the  absence 
of  pathogenic  bacteria.  If  death  occurs  the  toxic  or 
pathogenic  form  can  be  found  arid  isolated  from  the  organs 
and  tissues  of  the  animal  (see  anthrax). 

When  it  is  desired  to  examine  snow  or  ice  this  should 
be  melted  in  a  sterilized  flask  and  the  water  thus  obtained 
is  examined  as  above. 

The  number  of  bacteria  present  in  water  from  various 
sources  is  subject  to  the  greatest  variation.  Thus  spring 
water  may  be  sometimes  wholly  free  of  microorganisms, 
but  as  a  rule  the  number  is  less  than  50  per  c.  c.,  and  may, 
in  exceptional  case«,  contain  3,000  per  c.  c.  In  well 
waters  considerable  variation  has  been  observed,  but 
usually  the  number  is  less  than  500  per  c.  c.  The  water 
of  deep  wells  may  be  said  to  be  free  of  microorganisms. 
The  same  may  be  said  to  be  true  of  the  water  of  lakes. 
The  number  of  bacteria  present  in  river  water  varies  from 
a  few  hundred  to  as  many  thousand,  but  in  the  neighbor- 
hood of  large  cities  it  may  reach  hundreds  of  thousands 
per  c.  c.  In  Paris  the  river  water  in  the  wool-washing 
stations  has  been  shown  to  contain  from  12  to  40  millions 
per  c.  c. 

LABORATORY  WORK. — Make  plate  cultures  of  two 
samples  of  water — tap-water  and  well-water.  Also  Petri 
dishes  of  milk.  The  gelatin  tubes  are  inoculated  with 
milk  in  the  same  manner  as  with  water. 


—  76  — 


BACTERIOLOGICAL  EXAMINATION  OF  SOIL. 

The  collection  of  samples  of  earth  from  various  depths 
can  be  readily  accomplished  by  means  of  Fraenkel's  earth  - 
borer.  For  each  culture  experiment  a  definite  quantity 
of  the  soil  should  be  weighed  out,  or  a  measured  volume 
taken.  The  latter  is  the  simpler  procedure,  and  can  be 
done  with  a  Loffler  platinum  spoon  (1-50  c.  c.)  which 
serves  the  purpose  of  a  standard  volume. 

With  a  sterilized  Loffler  spoon  transfer  one  spoonful 
of  the  earth  to  a  tube  of  liquid  gelatin.  Mix  thoroughly 
with  a  sterilized  platinum  wire  and  then  make  an  Esmarch 
roll-tube.  The  soil  and  the  organisms  present  are  thus 
brought  into  perfect  contact  with  gelatin,  and  after  a  lapse 
of  a  few  days  colonies  develop.  These  can  be  readily 
counted,  and,  if  necessary,  with  the  aid  of  an  Esmarch 
roll-tube  counter.  The  kind  of  organisms — bacteria, 
mould?,  etc. — can  be  determined  by  the  study  of  the 
colonies,  and  by  further  culture  and  examination. 

In  this  wav  it  is  easy  to  determine  approximately 
the  number  and  kind  of  organisms  present.  Unfortunately 
this  method  is  not  adapted  for  the  detection  of  anaerobic 
bacteria  which  are  apparently  widely  distributed  in  the 
earth,  and  are  represented  by  the  well-known  bacilli  of 
tetanus,  malignant  oedema  and  symptomatic  anthrax. 
These  have  thus  far  been  obtained  only  by  indirect 
methods  from  the  soil.  Thus  animals  are  inoculated  with 
earth,  and  from  the  tissues  and  organs  after  death  the 
bacteria  are  isolated. 

The  surface  layers  of  soil,  to  a  depth  of  about  two  feet, 
are  exceedingly  rich  in  bacteria.  The  number  has  been 
found  to  vary  from  100,000  to  350,000,  and  may  even  reach 
several  million,  per  c.  c.  The  number  rapidly  decreases  with 
the  depth,  and  at  9  to  12  feet  the  soil  is  practically  sterile. 

LABORATORY  WORK. — Examine  three  samples  of  soil  by 
the  above  method. 


MEMORANDA. 


MEMORANDA. 


—  77  — 


BACTERIOLOGICAL  EXAMINATION  OF  AIR. 

To  determine  the  number  and  kind  of  bacteria  present 
in  the  air  is  a  problem  of  considerable  importance,  and  can 
be  accomplished  quite  satisfactorily  with  Hesse's  appara- 
tus. The  large,  wide  tube  is  sterilized,  nutrient  gelatin 
introduced,  and  a  large  Esmarch  roll-tube  then  made.  The 
tube  thus  prepared  is  connected  with  an  aspirating  bottle 
of  known  volume.  In  this  way  a  definite  volume  of  air 
can  be  drawn  through  the  apparatus.  The  bacteria  pres- 
ent in  the  air  are  deposited  on  the  moist  gelatin  walls  of 
the  tube,  and  subsequently  develop,  forming  colonies- 
These  are  counted  and  the  number  of  colonies  per  liter  of 
air  is  thus  ascertained.  The  kind  of  bacteria  present  can 
be  determined  in  the  usual  way. 

The  method  of  Petri,  though  somewhat  more  compli- 
cated, requires  less  time  and  gives  excellent  results.  The 
air  is  filtered  by  means  of  an  aspirator  or  air  pump  through 
a  tube  filled  with  sterilized  sand.  The  sand,  which  then 
contains  the  bacteria  originally  in  the  air,  is  transferred  to 
a  Petri  dish  containing  gelatin,  thoroughly  mixed,  and  set 
aside  to  develop. 

The  number  of  bacteria  present  in  the  open  air  is  very 
small  and  rarely  exceeds  3-4  per  liter.  Usually  the  num- 
ber is  much  less  than  this.  Spores  of  moulds  are  more 
abundant  in  the  air  than  are  bacteria.  Air  of  mid- ocean 
and  of  high  altitudes  is  practically  free  of  microorganisms. 


—  78  — 


PREPARATION  OF  BREAD  FLASKS. 

Moist  bread,  like  potatoes,  owing  to  its  slightly  acid 
acid  reaction,  is  an  excellent  medium  for  the  growth  of 
some  organisms,  especially  moulds. 

Prepare  some  dry  powdered  bread,  which  can  be  read- 
ily done  by  over-toasting  it  in  the  dry-heat  oven,  and  then 
crushing  or  pulverizing  the  dry  mass.  Keep  in  a  stoppered 
bottle. 

Clean,  plug,  and  sterilize  in  the  dry-heat  oven  six 
small  Erlenmeyer  flasks.  When  cool  cover  the  bottom  of 
the  flasks  to  a  depth  of  about  ^  inch  with  the  dry  powdered 
bread,  then  add  water  till  the  mass  becomes  thoroughly 
moist  and  soft.  Sterilize  in  steam  sterilizer  for  three  con- 
secutive days,  \  hour  each  day. 

Inoculate  the  bread  flasks  with  the  following  moulds  : 
Penicillium  glaucum. 
.Mucor  corymbifer. 

"      rhizopodiformis. 
Aspergillus  niger. 

"  flavescens. 

"  fumigatus. 

All  these  flasks,  except  the  first,  should  be  placed  in 
the  incubator  at  about  37°  C.,  for  24  to  36  hours.  They 
should  then  be  examined  for  the  characteristic  fruit  organs 
and  spores.  Transfer  a  portion  of  the  growth  to  a  watch- 
glass  containing  about  50  per  cent,  alcohol,  to  which  a 
drop  or  two  of  ammonium  hydrate  has  been  added.  When 
the  growth  becomes  moist,  transfer  a  portion  to  a  drop  of 
glycerine  on  a  slide.  Tease  out  the  specimen  thoroughly 
and  carefully  with  needles  or  pins.  Cover  with  a  cover- 
glass  and  examine  with  No.  7  objective  the  fruit  organs 
and  the  structure.  If  the  specimen  is  satisfactory  it  may 


MEMORANDA. 


MEMORANDA. 


—  79  — 

be  made  permanent  by  placing  a  ring  of  asphalt,  with  the 
aid  of  a  turn-table,  around  the  edge  of  the  cover-glass. 

Make  Petri  dishes  of  white  yeast;  Esmarch  roll  tubes 
of  red  yeast  and  of  black  yeast. 

Examine  baker's  or  brewer's  yeast  (Saccharomyces 
cerevisise)  in  hanging  drop  and  in  stained  preparations. 
Observe  the  form  and  structure  of  the  yeast  cell  and  the 
method  of  multiplication — by  budding. 


MEMORANDA. 


MEMORANDA. 


—  82  — 


PENICILLITJM  GLAUCUM. 

ONE    OF    THE    MOST    COMMON    GREEN    MOULDS. 

Origin. — Widely  distributed  in  the  air,  water,  soil. 
Color. — Green. 

Mycelium. — Consists  of  horizontally  arranged, 
straight  or  slightly  wavy,  jointed  mycelial  threads  from 
which  the  fruit  hyphge  rise  vertically. 

Fruit-organs. --The  ends  of  the  fruit  hyphae  are 
forked,  and  on  the  ends  are  the  intermediate  spore  bear- 
ers, or  "sterigmae,  also  sometimes  called  basidia.  Each  of 
these  in  turn  bears  a  row  of  spores  or  conidia,  so  that  the 
appearance  of  the  whole  is  that  of  a  brush. 

Gelatin  plates. — The  colonies  form  whitish  floccules  which  rapidly 
increase  in  size,  and  at  the  same  time  the  center  colors  green.  The  gelatin  is 
liquified  quite  early.  A  low  objective  will  show  the  above  chai'acteristics  of 
growth. 

Bread  flasks.— Show  a  low,  finely  flocculent  covering,  which  at  first  is 
white  but  soon  changes  to  a  distinct  green. 

Temperature.— Optimum    temperature  is   from  22 
to  26°  C.     Does  not  grow  at  the  temperature  of  the  body. 
Behavior  to  Gelatin.— Liquefies. 
Pathogenesis. — Has  no  effect  on  animals. 


Mi  ]V:CIMM?A. 


—  84  — 


MTJCOR,  CORYMBIFER.    Lichtheim. 

THE    MOST    COMMON    AND    WIDELY    DISTRIBUTED    MUCOR    IS    MUCOR 
MUCEDO,    OCCURRING    ON   EXCRETA,    ETC. 

Origin. — Is  of  rare  occurrence,  and  was  found  as  a 
contamination  on  bread-gelatin  plates.  Is  present  in  white 
bread,  and  has  been  found  in  the  ear-passages  of  man. 

Color. — Forms  a  snowy,  cotton-like  growth. 

Mycelium. — Loose,  wavy,  branching,  slender  mycel- 
ial  threads. 

Fruit-organs. — The  fruit  hyphae  branch  forming 
clusters  or  corymbs  which  terminated  with  spherical  or 
pear-shaped  sporangia.  Within  these  are  the  oval  or 
elongated  spores. 

Growth. — Rapid  and  extensive. 

Bread  flasks. — In  the  incubator  forms  a  white,  elevated,  cotton-like 
growth  which  soon  fills  the  flask. 

Temperature.— Grows  slow  at  ordinary  tempera- 
ture; best  at  37°  C. 

Pathogenesis. — Intravenous  injection  of  the  spores 
into  rabbits  produces  death  in  3  to  4  days.  The  kidneys, 
mesenteric  glands,  Peyer's  patches  contain  mycelial 
masses.  The  Peyer's  patches  are  swollen  and  ulcerated. 
Intraperitoneal  injections  produce  the  same  results. 
Dogs  are  immune. 


MEMORANDA. 


—  86  — 


MUCOR  RHIZOPODIFORMIS.    Lichtheim. 

Origin.— White  bread  kept  at  37°  0. 

Color.— At  first  white,  but  later  becomes  grayish. 

Mycelium. — The  mycelial  threads  are  colorless  and 
thicker  than  in  the  preceding  mucor,  and  are  not  jointed 
or  divided. 

Fruit-organs. — The  fruit  hyphae  occur  in  groups  or 
bunches,  which  adhere  to  the  nutrient  medium  by  means 
of  special  root  tufts.  The  large  sporangia  on  the  ends  of 
the  hyphae  contain  rounded  spores  which  are  larger  than 
those  of  the  preceding  organism. 

Growth. — Rapid. 

Gelatin  plates— Development  is  best  when  the  gelatin  is  made  with  bread 
infusion.  It  forms  a  coarse  grayish-black  mass  which  liquefies  the  gelatin. 

Bread  flasks. — The  growth  is  lower  than  that  of  M.  corymbifer,  and  is 
grayish,  owing  to  the  dark  colored  sporangia.  An  ethereal  or  aromatic  odor 
is  present. 

Temperature. — Slow  growth  at  12  to  15°,  but 
develops  best  at  37°  0. 

Behavior  to  Gelatin.— Liquefies. 

Pathogenesis. — Has  a  similar  effect  as  M.  corymbi- 
fer,  but  is  more  pathogenic. 


MEMORANDA. 


ASPERGILLUS  NIGER.    Van  Tieghem. 

Origin. — In  putrid  organic  substances ;  in  lungs  of 
birds. 

Color. — Black  or  dark  brown. 

Mycelium. — The  arrangement  is  much  the  same  as 
in  penicillium. 

Fruit-organs. — The  fruit  hyphse  are  swollen  or 
flask  or  club-shaped  at  the  end,  and  this  enlargement  is 
covered  willi  radially  arranged  minute  bottle-shaped 
bodies — the  intermediate  spore  bearers  or  sterigmse  from 
which  rows  of  spores  extend.  Sterigmee  divided. 

Growth.— Slow. 

Bread  flasks  —Forms  a  slow  growth  which  becomes  very  black. 

Temperature.— Its  optimum  is  about  35°  C. 

Pathogenesis. — Intravenous  injection  of  spores  in 
rabbits  is  not  followed  by  as  malignant  results  as  with  the 
next  two  forms. 


ASPERGILLUS  FLAVESCENS.     Wreden. 

Origin.— White  bread. 

Color. — At  first  whitish,  eventually  pale  yellow. 

Mycelium. — The  mycelial  threads  and  spores  are 
smaller  than  those  of  A.  niger. 

Fruit-organs.— The  club  shaped  ends  of  the  fruit 
hyphge  are  covered  with  sterigmae,  from  which  extend 
rows  of  spore?,  as  in  A.  niger. 

Growth.— Rapid. 

Bread  flasks. — Grows  best  on  bread.    Forms  a  yellowish,  low  growth. 

Temperature.— Optimum  about  28°  C.  Grows  well 
in  incubator. 

Pathogenesis. — Is  more  pathogenic  than  A.  niger, 
and  less  than  A.  fumigatus. 


MEMORANDA, 


—  90  — 


ASPERGIKLUS  FUMIGATUS.     Lichtheim. 

Origin. — White  bread.     In  the  air  passages  of  a  bird. 

Color.— Greenish  or  bluish  green  growth,  resembling 
very  much  that  of  penicillium. 

Mycelium. — About  same  as  preceding. 

Fruit-organs. — About  same  as  preceding,  but  spores 
only  about  one-half  as  large. 

Growth. — Is  best  on  bread  and  is  rapid. 

Bread  flasks.— The  growth  is  low  and  at  first  is  bluish  green,  but  when 
old  is  grayish  green. 

Temperature.— The  optimum  is  39-40°  C.  Can 
grow  at  the  ordinary  temperature. 

Pathogenesis. — Intravenous  injections  of  spores  in 
rabbits  and  dogs  produced  death  in  a  few  days.  Mycelia 
are  found  in  the  kidneys,  heart  muscle  and  other  muscles, 
and  occasionally  in  the  liver. 


MEMORANDA. 


—  92  — 


RED  YEAST. 

SEVERAL    RED     YEASTS    ARE     KNOWN.        THE    RED,     WHITE    AND    BLACK 
YEASTS    ARE    NOT    TRUE    YEAST-PLANTS. 

Origin. — Very  common  in  air. 
Color.— Bed  or  pink. 

Form. — Round  [or  oval  cells  with  granular  proto- 
plasm which  stains  irregularly.  Multiplies  by  budding- 
distinction  from  bacteria. 

Motility. — None. 

Sporulation. — None. 

Anilin  Dyes. —Stain  readily. 

Growth. — Abundant,  though  somewhat  slow. 

Gelatin  Plates. — Colonies  are  small,  round,  elevated,  moist  and  pink- 
colored. 

Stich  Cultures. — Growth  absent  from  the  lower  part  of  the  tube.  Spreads 
slowly  over  the  surface,  forming  a  thick,  moist,  bright  red  covering. 

Streak  Cultures.  —  On  agar,  develops  in  a  few  days  as  a  thick,  slimy, 
spreadftig,  pink-colored  growth.  On  potatoes,  forms  the  same  pigment. 

Temperature. — Grows  best  at  ordinary  temperature. 
Behavior  to  Gelatin.— Does  not  liquefy. 
Aerogenesis.— Does  not  produce  alcohol. 
Pathogenesis.— No  effect  on  animals. 


MEMORANDA. 


—  94  — 


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MEMORANDA. 


—  95  — 


PREPARATION     OF     NUTRIENT    BOUILLON 
AND  OF  NUTRIENT  AGAR. 

Place  500  g.  of  chopped  lean  beef  into  a  clean  1^  or 
2-litre  flask  ;  add  1000  c.  c.  of  tap-water  and  insert  a  cotton 
plug  into  the  neck  of  the  flask.  Shake  the  flask  repeatedly 
during  the  next  15  or  30  minutes,  then  immerse  in  a  water- 
bath  containing  boiling  water,  and  heat  for  15  or  30  min- 
utes. Filter  through  muslin,  and  to  the  filtrate  or  meat 
extract  thus  obtained  add 

10  g.  of  dry  peptone, 
5  g.  of  common  salt. 

Warm  in  the  water-bath  for  some  minutes,  then  render 
slightly  alkaline  by  cautious  addition  of  saturated  sodium 
carbonate  solution.  Continue  heating  in  a  water-bath, 
with  occasional  shaking,  for  f  to  1  hour,  then  filter  through 
a  plaited  filter— bouillon.  The  bouillon  thus  prepared 
should  be  (1)  perfectly  clear;  (2)  should  be  slightly 
alkaline  in  reaction,  and  (3)  should  not  coagulate  or  cloud 
on  heating. 

Take  100  c.  c.  of  the  bouillon  and  fill  into  sterilized 
plugged  test-tubes,  using  a  small  funnel,  to  a  depth  of 
1 — 1^  \i\c\\e'&= ordinary  bouillon. 

To  100  c.  c.  of  the  bouillon  add  two  per  cent,  of 
glucose,  warm  till  dissolved,  then  fill  likewise  into  test- 
lubes— glucose  bouillon. 

To  the  remainder  of  the  bouillon  (800  c.  c.)  add  H  to  2 
per  cent,  of  agar,  previously  cut  up  into  very  small  pieces. 
Place  on  a  wire  gauze  and  heat  at  just  below  the  boiling 
point  (to  prevent  frothing)  for2or3hours,  till  the  agar  has 
completely  dissolved.  Then  turn  the  flame  low  and  con- 
tinue heating  for  1  or  2  hours,  when  the  suspended  matter 
largely  subsides  to  the  bottom.  Then  filter  by  decaritation 
9 


—  96  — 

through  a  layer  of  cotton  placed  in  the  neck  of  the  funnel, 
and  it'  the  filtrate  is  not  fairly  clear,  heat  it  to  the  boil- 
ing-point and  filter  again  through  the  same  cotton  filter. 
The  nutrient  agar  thus  obtained  should  be  (1)  transparent 
or  semi-transparent ;  (2)  slightly  alkaline  in  reaction,  and 
(3)  should  not  coagulate  or  cloud  on  heating. 

To  100  c.  c.  of  the  filtered  agar  add  6  to  7  c.  c.  of  pure 
glycerine,  mix  thoroughly;  lest  the  reaction  again  to  see  if 
alkaline,  and  then  fill  into  sterilized  test-tubes=^///cer^e 
agar. 

To  100  c.  c.  of  the  filtered  agar  add  2  per  cent,  of  glu- 
cose;  warm  till  dissolved,  then  fill  into  sterilized  test- 
tubes  to  a  height  of  about  1|  to  2  \\\U\\QS= glucose  ayar. 

Transfer  the  remainder  of  the  agar  to  sterilized  test- 
tubes^  ordinary  agar. 

Sterilize  the  bouillon  and  agar  tubes  l>y  heating  in 
steam  sterilizer,  4-  hour  each  day,  for  three  consecutive 
days.  At  the  end  of  the  third  heat  place  the  agar  tubes, 
while  still  liquid,  in  a  slightly  inclined  position,  so  that 
the  agar  reaches  to  within  1  to  H  inches  of  the  plug,  and 
allow  it  to  solidify. 

Instead  of  preparing  an  extract  of  fresh  meat  it  is 
sometimes  more  convenient  to  employ  commercial  meat 
extract,  such  as  Liebig's.  In  that  case  2.5  g.  of  Liebig's 
extract  with  the  usual  amount  of  peptone  and  common 
salt  is  added  to  100U  c.  c.  of  water.  To  this  solution  or 
bouillon  the  ordinary  proportion  of  agar  or  gelatin  is 
added,  and  the  nutrient  media  are  otherwise  prepared  in 
exactly  the  same  manner  as  already  given. 

When  a  perfectly  transparent  agar  is  desired  it  is,  as  a 
rule,  necessary  to  filter  though  paper.  Thiscan  be  accom- 
plished most  rapidly  by  placing  a  filter-stand  with  funnel 
and  plaited  filter,  slightly  moistened,  in  a  steam  sterilizer. 
When  the  funnel  is  thoroughly  heated  the  boiling  agar 
solution  is  transferred  to  the  filter. 


MEMORANDA. 


MEMORANDA. 


—  97  — 


PATHOGENIC  BACTERIA. 

By  the  application  of  the  gelatin  plate  method  it  is 
possible  to  readily  separate  a  given  organism  from  other 
forms  which  may  be  present  and  thus  obtain  a. pure  cul- 
ture. The  isolated  colony  as  it  develops  on  a  plate  fur- 
nishes the  first  pure  cultivation  since  it  is  derived  from  a 
single  micro-organism.  Transplantations  made  from  a 
colony,  if  made  with  proper  piecautions,  in  turn  yield 
pure  cultures  or  growths  containing  but  a  single  species. 
Tube  cultures  can  thus  be  made  in  gelatin,  bouillon,  agar, 
blood  serum,  potato,  etc.,  and  where  it  is  desired,  as  in  the 
study  of  chemical  products  of  bacteria,  flask  cultures  can 
be  made. 

It  is  evident  that  in  order  to  demonstrate  that  a  given 
bacterium  is  the  cause  of  a  certain  fermentation,  or  of  the 
production  of  some  pigment  or  of  phosphorescence,  etc.,  it 
is  necessary  that  it  should,  first,  be  isolated  and  obtained 
in  pure  cultures,  and  that,  second,  pure  cultures  of  the 
organism  grown  under  the  same  or  similar  conditions, 
should  give  rise  to  the  original  phenomena — the  produc- 
tion of  the  same  fermentation,  pigment,  phosphorescence, 
etc.  Having  thus  demonstrated  that  a  given  organism  is 
the  cause  of  certain  changes  it  does  not  follow  that  this 
organism  has  the  exclusive  power  to  do  so.  Thus,  in  alco- 
holic fermentation  the  yeast  plant  is  commonly  said  to  be 
the  cause,  but  a  large  number  of  different  species  of  yeasts 
are  known  which  have  this  power,  and  not  only  the 
yeasts  but  many  bacteria  possess  similar  properties. 
Again  a  considerable  number  of  bacteria  have  been  shown 
to  be  capable  of  inducing  acetic,  lactic,  butyric  acid  fermen- 
tations, the  ammoniacal  and  hydrogen  sulphide  fermen- 
tations of  urine,  the  phosphorescence  of  sea-water,  etc. 

The  most  that  can  be  said  of  a  given  organism  which 


—  98  — 

induces  a  certain  change,  therefore,  is  that  it  is  the  cause 
in  that  particular  instance.  The  possibility  of  other 
organisms  giving  rise  to  the  same  changes,  or  effect,  or 
chemical  products,  must  be  conceded,  and  the  demonstra- 
tion of  the  relations  of  an  organism  to  such  a  change  rests 
with  the  proof  that  it  is  a  cause. 

Just  as  there  are  organisms  which  induce  changes 
in  dead  animal  or  vegetable  matter,  there  are  others  which 
are  capable  of  inducing  similar  changes  in  living  animals 
and  plants,  thus  living  at  the  expense  and  frequently  to 
the  detriment  of  the  host.  The  infectious  diseases  in  man, 
animals  and  plants,  possess  as  an  essential  characteristic 
the  property  of  transmissibility.  They  are  the  result,  first, 
of  infection  that  is  the  entrance  of  a  specific  micro organ- 
ism, and  second,  of  intoxication  due  to  the  poisonous  pro- 
ducts elaborated  by  the  microorganism.  Poisonous  chem- 
ical compounds  may  produce  the  symptoms  and  the 
changes  observed  in  an  infectious  disease.  They  are 
the  cause  of  those  symptoms  and  changes,  but  they 
are  not  the  cause  of  disease,  since  the  symptoms  and 
changes  thus  obtained  are  not  transmissible  from  one 
individual  to  another.  Chemical  substances  have  no 
power  of  multiplication  and  the  effect  observed  is,  there- 
fore, directly  proportional  to  the  amount  of  the  chemical 
compound  introduced.  Microorganisms,  however,  have  the 
power  of  multiplication,  and  the  introduction  of  a  minute 
amount,  even  a  single  cell,  may  bring  about  entirely  dis- 
proportionate results.  The  invading  organism  is  therefore 
the  cause  of  the  disease  since  it  imparts  the  characteristic 
property  of  transmissibility,  and,  through  the  action  of  its 
chemical  products,  produces  the  symptoms  and  effects  of 
that  disease. 

In  order  to  positively  demonstrate  the  causal  relation 
of  a  microorganism  to  a  given  disease,  it  is  necessary  to 
meet  the  following  requirements,  commonly  known  as  the 
four  rules  of  Koch  : 

(1.)  The  organism  must  be  present  in  all  cases  of 
that  disease. 


MEMORANDA. 


MEMORANDA. 


—  99  — 

(2.)  The  organism  must  be  isolated  and  obtained  as 
an  absolutely  pure  culture. 

(3.)  The  pure  culture  of  the  organism  when  intro- 
duced into  susceptible  animals  must  produce  the  disease. 

(4.)  In  the  disease  thus  produced  the  organism  must 
be  found  distributed  the  same  as  in  the  natural  disease. 

To  these  four  requirements,  a  fifth  may  be  added, 
namely:  That  the  chemical  products  of  the  organism  must 
produce  the  characteristic  symptoms  and  effects  of  that 
disease. 

The  demonstration  of  the  constant  presence  of  an 
organism  in  a  disease  is  accomplished  by  hanging-drop 
examination,  stained  cover-glass  preparation,  or  by  stain- 
ing sections  of  tissues  and  organs.  Frequently  the  direct 
detection  of  the  organism  is  difficult  owing  either  to  its 
scarcity  or  to  the  absence  of  definite  characteristics.  In 
such  cases  artificial,  culture  or  animal  experiment  will 
prove  the  presence  of  the  organism. 

The  mere  fact  that  an  organism  is  constantly  present 
in  a  given  disease  does  not  prove  that  it  is  the  cause  of 
that  disease.  It  certainly  is  strong  presumptive  evidence 
that  the  organism  does  bear  a  causal  relation  to  that  dis- 
ease, but  at  the  same  time  the  possibility  must  be  admitted 
that  it  may  be  an  accompaniment,  or  even  a  consequent 
of  ihat  disease.  To  complete  the  chain  of  evidence  it  is 
necessary,  therefore,  to  obtain  the  organism  in  a  pure 
culture,  and,  inoculation  of  animals  with  such  cultures 
must  reproduce  the  disease. 

The  isolation  of  the  organism  and  the  preparation  of 
pure  cultures  is  accomplished  by  the  gelatin  plate  method 
or  its  modifications.  The  isolated  colony  which  develops 
on  a  plate  is  derived  from  a  single  cell,  and  is,  therefore,  a 
pure  culture.  Transplantations  from  the  colony,  when 
properly  made,  into  tubes  of  gelatin,  agar,  or  bouillon,  in 
turn  are  pure  cultures.  Subsequent  transplantations  from 
tube  to  tube  can  be  made  as  often  as  may  be  desired,  or  as 
may  be  necesssary.  Each  growth  thus  obtained  is  called 


—  100  — 

a  generation.  In  many  cases,  as  in  tuberculosis,  anthrax, 
and  in  hog  cholera,  the  organisms  have  thus. been  carried 
through  several  hundred  consecutive  generations  without 
impairment  of  pathogenic  properties.  In  other  instances, 
as  in  glanders,  the  organism  does  not  find  in  our  artificial 
media  the  conditions  favorable  for  its  growth  and  as  a 
result  it  undergoes  a  physiological  alteration  so  that  the 
cultures  become  less  and  less  active  till  finally  they  cease 
to  have  any  effect  on  animals.  This  change  in  the  physi- 
ological properties  of  an  organism — known  as  attenuation 
—is  frequently  accompanied  by  a  corresponding  decrease 
in  the  vitality  of  the  growth  so  that,  when  the  virulence  is 
wholly  lost,  the  culture  soon  dies  out.  Sometimes,  how- 
ever, the  organism  adapts  itself  to  the  artificial  media  and 
continues  to  grow  although  with  diminished  pathogenic 
properties. 

The  above  four  rules  have  been  fully  complied  with 
in  a  large  number  of  infectious  diseases.  In  others  the 
first  two  rules  ;ire  s;itij-fied  bill  the  third  is  nor,  owing  to 
the  difficulty  of  obtaining  a  susceptible  animal.  Ay:ain,  the 
first  rule  maybe  ihoonly  one  complied  with,  as  in  leprosy, 
where  the  isolaiio'i  of  the  organism  has  not,  thus  far, 
been  unquestionably  successful.  And  again  a  large 
number  of  infectious  diseases  remain,  in  which  even  the 
presence  of  a  specific  organism  has  not  been  definitely 
shown. 

Although  many  of  the  infectious  diseases  have  been 
shown  to  be  due  to  bacteria,  it  must  not  be  forgotten  that 
other  low  forms  of  plant  and  animal  life  possess  sim- 
ilar properties.  Thus  there  are  infectious  diseases  due  to 
fungi  and  also  such  as  are  due  to  animal  parasites — sporo- 
zoa,  etc. 


MEMORANDA. 


MEMORANDA. 


—  101  — 


METHODS  OF  INFECTION. 

1. — Cutaneous  application. 
2. — Subcutaneous  application. 

In  mice  and  rats  this  can  best  be  done  on  the  back, 

over  the  root  of  the  tail. 
3. — Subcutaneous  injection. 

The    Koch    syringe    is    commonly    used.      Pravaz 

syringe. 
4. — Intravenous  injection. 

The  large  veins  in  the  ears  of  rabbits  are  frequently 

used  ;  also  the  jugular  and  femoral  veins. 
5. — Intraperitoneal  injection. 
6. — Intrapleural  injection. 

7. — Injection  into  the  anterior  chamber  of  the  eye. 
8.— Infection  along  respiratory  tract. 

(a)  Inhalation,     (b)  Injection  into  the  trachea. 
9. — Infection  of  alimentary  canal. 

(a)  With  food  or  drink. 

(b)  Through  a  stomach-tube — the   contents  of  the 

stomach  are  previously  rendered  alkaline. 

(c)  Intraduodenal  injection. 

The  inoculation  of  animals  with  pure  cultures  of 
microorganisms  must  be  made  with  rigid  precautions  to 
prevent  the  introduction  of  foreign  organisms.  At  the 
site  of  inoculation  the  hair  must  be  carefully  cut  away ; 
the  exposed  skin  is  then  well  washed  with  alcohol,  and 
finally  is  thoroughly  moistened  with  mercuric  chloride 
(1-1000).  The  instruments  employed  in  making  the  inoc 
illations,  as  knives,  scissors,  forceps,  lance,  wire,  etc.,  must 
be  sterilized  in  a  flarne  shortly  before  use.  The  Koch 
syringe  is  sterilized  in  the  dry  oven.  After  having  used 
the  instruments  they  are  at  once  sterilized. 


—  102  — 

In  working  with  the  pathogenic  microorganisms  the 
student  must  specially  observe  the  utmost  precaution 
against  personal  infection.  The  rule  to  sterilize  every 
instrument  shortly  before,  and  immediately  after  use, 
before  it  has  left  the  hands,  must  be  strictly  attended  to. 
Direct  contact  of  the  hands  with  infectious  matter  must 
be  carefully  avoided,  and  when  such  contact  has  taken 
place  prompt  disinfection  must  be  resorted  to.  On  no 
account  must  lead  pencils  or  glass  rods  be  held  in  the 
mouth,  or  labels  moistened  on  the  tongue.  Gelatin  or  agar 
plates  must  be  set  aside  for  some  hours  in  mercuric  chlo- 
ride (1-1000).  Old  tube  cultures  are  best  sterilized  by 
heating  in  the  steam  sterilizer  for  about  one-half  hour.  If 
by  accident  infectious  matter  is  dropped  on  the  table  or 
floor  it  must  at  once  be  covered  with  mercuric  chloride. 
At  the  close  of  the  day's  work  the  table  must  be  well 
washed  with  the  mercury  solution,  and  the  hands 
thoroughly  disinfected. 


MEMORANDA. 


MEMORANDA. 


—  103  — 


POST-MORTEM  EXAMINATION. 

Demonstration  of  post-mortem  on  guinea-pig  that 
died  aftor  subcutaneous  inoculation  with  the  anthrax 
bacillus. 

The  animal  is  placed  on  a  board  ;  the  feet  are  extended 
and  tacked  or  nailed  down.  The  hair  over  the  abdomen 
and  thorax  is  moistened  thoroughly  with  a  cloth  soaked 
in  mercuric  chloride.  With  a  pair  of  sterilized  forceps  the 
skin  over  the  lower  part  of  the  abdomen  is  raised  and  a 
slight  transverse  nick  is  made  with  sterilized  scissors. 
Into  the  opening  thus  made  the  lower  blade  of  the  scis- 
sors is  introduced  and  an  incision  is  made  along  the  median 
line  to  the  neck.  While  making  the  incision  the  skin  is 
kept  raised  by  means  of  the  forceps  to.  avoid  cutting 
through  the  abdominal  or  thoracic  walls.  At  each  end  of 
this  incision  lateral  cuts  are  made  in  the  direction  of  the 
extremities,  and  the  two  flaps  of  skin,  thus  prepared,  are 
carefully  reflected,  thus  exposing  the  entire  abdominal 
and  thoracic  walls.  The  condition  of  the  subcutaneous 
tissue,  of  the  abdominal  walls,  of  the  blood-vessels  and 
the  presence  or  absence  of  oedema,  gas,  etc.,  should  be 
noted. 

The  scissors  and  forceps  are  sterilized,  and  when  cool 
a  similar  incision  is  made  into  the  abdominal  wall  and 
extended  through  the  cartilages  of  the  ribs  to  the  neck. 
Special  care  must  be  taken  to  prevent  cutting  into  the 
intestines  or  internal  organs.  Lateral  cuts  are  made  as 
before,  and  after  nicking  the.  ribs  on  the  inside  of  the  thorax 
close  to  the  vertebral  column,  the  entire  abdominal  and 
thoracic  walls  can  be  reflected,  thus  exposing  to  view  all 
the  internal  organs.  The  condition  of  the  abdominal  and 
thoracic  cavities  should  be  observed  ;  also  the  appearance 
of  the  peritoneum,  liver,  spleen,  kidneys,  heart,  lungs,  etc. 

10 


—  104  — 

With  re-sterilized  forceps  and  scissors  (lie  spleen, 
kidneys,  liver,  etc.,  should  be  removed  to  sterilized  Petri 
or  Esmarch  dishes  and  can  be  used  for  subsequent  exam- 
ination. 

In  making  post-mortem  examinations  the  utmost  care 
must  be  taken  to  prevent  the  introduction  of  foreign 
microorganisms,  and  at  the  same  time  to  prevent-  scatter- 
ing any  infectious  matter,  from  the  animal.  For  that 
reason  the  hair  on  the  skin  is  thoroughly  moistened  to 
prevent  it  from  flying  about  or  entering  the  opening  in  the 
body.  The  forceps,  knives  and  scissors  must  be  sterilized 
in  a  flame  for  each  separate  incision.  When  blood  or 
pieces  of  tissue  adhere  to  the  instruments,  they  should  not 
be  placed  at  once  into  the  flame,  otherwise  the  sudden 
heating  will  cause  the  material  to  spurt  and  scatter  about. 
To  avoid  this  the  material  should  first  be  dried  by  holding 
I  he  instruments  close  to  the  flame.  This  precaution  should 
also  be  observed  when  sterilizing  wires  which  are  covered 
with  gelatin. 

LABORATORY  WORK,  WITH  ASTHKAX  TISSUE. 

Isolation  of  the  bacillus  in  pure  culture. — The  bacil- 
lus of  anthrax  which  is  present  in  the  blood,  tissues  and 
organs  of  the  guinea  pig,  must  be  isolated  and  obtained  in 
pure  culture.  This  can  be  readily  accomplished  by  the 
gelatin  plate  method.  For  this  purpose  a  small  piece  of 
liver,  about  half  the  size  of  a  grain  of  wheat,  is  cut  off  with 
a  sterilized  pair  of  scissors.  The  piece  of  tissue  is  placed 
on  the  loop  of  a  sterilized  platinum  wire  and  transferred 
to  a  tube  of  liquefied  gelatin.  By  rubbing  the  piece 
against  the  walls  of  the  tube  with  the  wire  the  blood  can 
be  squezed  out  and  the  oigani-m  piesent  is  thus  spread 
throughout  the  gelatin.  From  this  tube,  which  is  No.  1, 
transfers  are  made  in  the  usual  manner  to  tube  No.  2,  and 
from  this  to  tube  No.  3.  Gelatin  plates  are  then  made  in 
the  usual  manner,  and  set  aside  for  two  or  three  days  to 
develop. 


MEMORANDA. 


MEMORANDA. 


—  105  — 

When  the  colonies  develop  their  form  should  be  care- 
fully studied  as  it  is  very  characteristic,  and,  if  possible, 
impression  preparations  should  b6  made  from  the  surface 
colonies  and  stained  with  methylene  blue. 

As  the  colony  is  a  pure  culture  of  the  anthrax  bacillus, 
transplantations  to  tubes  in  turn  yield  pure  cultures.  Make 
a  stich  culture  in  gelatin  and  a  streak  culture  on  inclined 
agar.  This  latter  is  made  by  simply  drawing  the  end  of 
the  platinum  wire  along  the  middle  on  the  surface  of  the 
agar.  The  agar  tube  is  placed  in  the  incubator  at  37  to 
39°  G.  for  one  or  two  days,  then  removed  and  examined. 

Another  agar  tube  is  liquefied  and  -J  to  1  drop  of  cal- 
cium hydrate  is  added,  thoroughly  mixed,  and  the  tube  is 
then  set  aside  in  an  inclined  position  till  the  agar  solidifies. 
Then  make  a  streak  culture  on  this  Ca  (OH)2-agar,  and 
set  it  aside  to  develop  in  the  incubator. 

With  the  pure  cultures  of  the  anthrax  bacillus  thus 
obtained  the  student  can  inoculate  a  number  of  white 
mice,  white  rats  and  rabbits,  and  in  these,  after  death,  the 
organism  can  in  turn  be  detected  and  isolated.  In  this 
way  each  one  has  an  opportunity  to  demonstrate  all  four 
rules  of  Koch  with  reference  to  anthrax,  thus  proving  that 
the  anthrax  bacillus  is  the  cause  of  the  disease. 

MICROSCOPICAL  EXAMINATION. 

Hanging-drop. — Take  a  clean  f -inch  cover-glass  and 
pass  it  once  through  the  flame.  Transfer  a  small  drop  of 
sterile  bouillon  to  the  cover-glass  and  then  add  to  it,  with  a 
sterilized  wire,  a  minute  amount  of  the  heart-blood. 
Apply  the  concave  slide,  ringed  with  vaseline,  and 
examine  the  hanging-drop,  thus  prepared,  with  the  No.  7 
objective.  Study  the  characteristics  of  the  anthrax 
bacillus  as  it  exists  in  the  blood,  and  compare  its  size  with 
that  of  the  blood-cell.  Then  label  the  slide  and  set  aside 
in  the  incubator  for  24  hours.  Examine  the  slide 
on  the  following  day  and  observe  the  formation  of 
threads,  of  sporogenic  granules  and  possibly  of  spores. 


—  106  — 

Finally  make  permanent  stained  mounts  of  these  threads 
by  transferring  a  small  portion  of  this  drop  culture  to  a 
minute  drop  of  water  on  a  clean  cover-glass  ;  spread,  dry, 
fix  and  stain  the  preparation  in  the  usual  manner. 

Stained  preparations. — Place  about  two  dozen  clean 
cover-glasses  on  the  lid  of  a  slide  box.  Pick  up  a  piece  of 
the  spleen,  kidney  or  liver  in  a  pair  of  forceps,  and  while 
holding  the  cover-glass  down  with  another  pair  of  forceps, 
lightly  streak  the  cut  surface  of  the  organ  over  the  cover- 
glass.  A  very  thin  and  even  film  is  desirable.  In  this 
way  streak  all  the  cover-glasses,  then  allow  them  to  dry 
in  the  air  and  fix  cautiously  by  passing  once  or  twice 
through  the  flame.  These  cover- glasses  are  commonly 
known  as  streak  preparations. 

Stain  some  of  the  fixed  cover-glasses  will)  simple  ani- 
lin  dyes,  as  gentian  violet  or  i'uchsine ;  examine  and 
study  the  specimens  carefully  and  make  permanent  prep- 
arations. The  remainder  of  the  cover-glasses  will  serve 
for  double-staining  by  Gram's  method. 

Gramas  method. — This  excellent  method  for  demon- 
strating the  presence  of  certain  bacterin,  as  anthrax,  in  the 
fluids  and  tissues  of  the  body  is  based  upon  the  fact  that 
the  protoplasm  of  the  bacterial  cell  when  stained  with 
anilin  water-gentian  violet,  and  then  treated  with  iodine 
forms  a  difficultly  soluble  compound.  By  proper  exposure 
to  a  solvent  the  dye  can  now  be  removed  from  the  entire 
cover-glass,  but  not  from  the  bacterial  cell.  The  deeply 
stained  violet  rods  lie  on  a  colorless  back-ground,  which 
on  treatment  with  a  contrast  color,  as  eosine  or  picro  car- 
mine, becomes  stained  a  light  pink.  The  method  is  as 
follows : 

A  solution  of  anilin  water-gentian  violet  is  first  pre- 
pared. Anilin  oil  is  placed  in  a  test-tube  to  a  depth  of 
about  half  an  inch.  The  tube  is  then  filled  with  water, 
closed  with  the  thumb  and  thoroughly  shaken  in  order  to 
obtain  a  saturated  aqueous  solution  of  anilin.  The  liquid 
is  then  passed  through  a  small  filter  and  collected  in 


MEMORANDA. 


MEMORANDA. 


—  107  — 

another  test'- tube.  The  filtrate  should  be  perfectly  clear, 
not  cloudy.  To  the  anilin  water  thus  obtained  a  satu- 
rated alcoholic  solution  of  gentian  violet  is  added  till  the 
iluid  is  deeply  colored. 

Some  of  the  anilin  water-gentian  violet  thus  pre- 
pared is  poured  out  into  a  watch-glass.  A  streak  cover  - 
glass  preparation  of  anthrax  is  now  carefully  fixed  in  tlie 
flame.  Care  must  be  taken  not  t.o  over-heat  the  specimen, 
as  the  anthrax  bacillus  when  over-heated  does  not  stain 
satisfactorily.  The  fixed  cover-glass  is  then  placed 
between  the  thumb  and  forefinger,  with  the  specimen  side 
down,  and  carefully  dropped  upon  the  surface  of  the  stain 
in  the  watch-glass.  It  is  then  allowed  to  float  on  the  dye 
for  10  to  15  minutes.  Sometimes  it  is  necessary  to  warm 
the  dye  on  the  radiator  or  on  an  iron  plate  in  order  to 
obtain  a  rapid  and  intense  stain.  The  cover-glass  is  then 
picked  up  with  the  forceps,  thoroughly  washed  with 
water,  and  immersed  in  a  solution  of  iodine  in  potassium 
iodide.  This  is  made  by  dissolving  2  g.  of  potassium 
iodide  and  1  g.  of  iodine  in  300  c.  c.  of  distilled  water. 
The  specimen  is  allowed  to  remain  in  the  iodine  for  ^  to 
1  minute,  or  even  several  minutes.  Care  must  be  taken 
not  to  expose  too  long  to  the  action  of  iodine,  as  it  tends 
to  contract  the  protoplasm  into  granules.  The  cover-glass 
is  then  removed  from  the  iodine,  washed  with  water,  and 
moved  about  in  a  watch  glass  of  strong  alcohol,  to  which, 
if  necessary,  a  drop  of  acetic  acid  may  be  added,  to  assist 
the  decoloration.  From  time  to  time  the  cover-glass 
should  be  washed  with  water  and  examined  with  No.  7 
objective  to  ascertain  the  progress  in  decoloration.  When 
finally  a  colorless  back-ground  is  obtained  for  the  deeply 
stained  violet  bacilli  the  washing  in  alcohol  is  discontin- 
ued. The  cover-glass  is  then  washed  with  water  and 
stained  with  dilute  eosine  for  ^  to  ^  minute.  The  eosine 
is  an  acid  anilin  dye,  and  therefore  stains  the  protoplasm 
of  cells,  nuclei,  etc.,  but  not  bacteria.  Care  must  be  taken 
not  to  overstain  the  preparation  with  eosine,  as  it  would 


—  108  — 

tend  to  diminish  the  sharp  contrast  that  is  desired.  The 
specimens  after  staining  with  the  eosine  is  thoroughly 
washed  with  water  and  examined  under  the  microscope. 
It  should  show  the  deeply  stained  violet  bacilli  on  a  light 
pink  back-ground. 

Weigert's  picro-carmine  solution,  or  Bismarck  brown, 
can  be  also  used  for  contrast  colors.  The  Gram's  method 
is  applicable  to  many  pathogenic  bacilli  and  to  most 
micrococci.  A  notable  exception  among  the  latter  is  the 
gonococcus.  The  other  important  organisms  that  do  not 
stain  with  this  method  are  the  bacillus  of  typhoid  fever, 
of  Asiatic  cholera,  of  glanders,  of  chicken  cholera,  of 
rabbit  septicaemia;  also  Friedlaender's  pneumo-bacillus, 
and  the  spirillum  of  recurrent  fever. 

The  following  synopsis  of  the  staining  methods  for 
streak  preparations  will  be  of  service: 

Cover  glass  preparation. 

Air-dried. 

3  x  through  tlame. 


Simple  slain  :  Gram?s  stain  : 

Dilute  anilin  dye  Anilin  water  gentian    violet 

(i  to  |  min.).  (10  10  15  min.;    if  hot,  2  to 

Water  (and  examine).         5  inin.). 
Air-dried.  Water. 

Canada  balsam.  Iodine  in    potassium    iodide 

(|  to  1  to  3  min.). 
Water. 
Alcohol. 

Water  (and  examine). 
Contrast    color     (eosine     or 

picrocarmine,  few  sec.). 
Water  (and  examine). 
Air  dried. 
Canada  balsam. 

The  hanging-drop  examination  and  the  streak  prepa- 
rations  stained    by   the   simple   and   double    method    as 


MEMORANDA, 


MEMORANDA. 


—  109  — 

described,  serve  the  purpose  of  demonstrating1  the  pres- 
ence of  the  anthrax  bacillus  in  the  different  organs  and 
tissues  of  the  body.  The  form,  size,  etc.,  of  the  bacillus 
found  under  these  conditions  should  be  compared  with  the 
growth  of  the  organism  in  pure  cultures  in  different 
media.  For  this  purpose  make  hanging-drop  examina- 
tions and  permanent  simple  stains  of  the  bacillus  grown 
in  the  stich  culture  in  gelatin,  on  ordinary  agar,  and  on 
calcium  hydrate  agar.  The  preparation  of  impression 
cover-glasses  of  the  anthrax  colonies  and  simple  stains  of 
bouillon  hanging-drop  culture  have  been  mentioned. 

Double  stain  for  spores. — The  growth  of  the  anthrax 
bacillus  on  calcium  hydrate  agar  when  examined,  as 
mentioned  above,  in  hanging-drop  will  show  the  presence 
of  an  abundance  of  bright,  highly  refracting  oval  bodies, 
or  spores,  which  may  be  observed  free  and  also  within  the 
parent  cell.  Simple  stains  of  this  growth  with  fuchsine, 
etc.,  will  show  the  bacilli  deeply  stained,  whereas  the 
spores  remain  colorless.  This  is  undoubtedly  due  to  the 
dense  impenetrable  wall  which  surrounds  the  spores  and 
prevents  the  dye  from  passing  into  the  spore,  as  well  as  to 
a  special  composition  of  the  spore  contents.  By  proper 
treatment  with  strong  anilin  dyes  it  is  possible  to  force 
the  stain  into  the  spore.  Once  within  the  spore  it  is  as 
difficult  to  remove  the  dye  as  it  was  to  cause  it  to  enter. 
By  suitable  decoloration  it  is,  therefore,  possible  to  remove 
the  stain  from  everything  on  the  cover-glass,  except  from 
the  spores.  Then,  on  the  application  of  a  contrast  color  the 
specimens  will  show  a  bright  red  spore  within  a  blue 
bacillus.  The  method  of  double  staining  of  spores  is  as 
follows : 

The  cover-glass  preparation  from  the  calcium  hydrate 
agar  is  dried  in  the  air  and  fixed  in  the  usual  manner. 
The  cover-glass  is  held  in  the  forceps,  in  the  left  hand, 
with  the  specimen  side  up  and  covered  with  a  solution  of 
carbolic  fuchsine.  This  is  held  over  a  Bunsen  flame,  so 
that  vapors  are  given  off  from  the  liquid.  Active  ebullition 


—  110  — 

should  be  avoided.  From  time  to  time  the  liquid  which 
is  lost  by  evaporation  is  replaced  by  a  fresh  addition  of  the 
carbolic-fuehsine,  and  under  no  condition  should  the  dye 
be  allowed  to  dry  down  on  the  cover-glass.  Best  results 
in  heating  are  obtained  with  the  flame  turned  low,  so  that 
it  is  not  over  two  inches  high.  After  heating  the  speci- 
men in  this  manner  for  two  or  three  minutes  the  stain  is 
thoroughly  washed  off  with  water  and  the  cover-glass 
examined  wiih  the  No.  7  objective.  Colorless  spores 
should  no  longer  be  visible,  but  everything  should  be 
stained  a  deep  red.  If  the  spores  are  not  colored  the  heat- 
ing with  carbolic-fuchsine  is  repeated  until  they  become 
stained.  The  cover-glass  may  be  floated  on  hot  carbolic- 
fuchsine  in  an  Esmarch  dish  for  ^  to  1  hour. 

The  cover  glass  having  deeply  stained  spores  is  then 
moved  about  in  dilute  alcohol,  and,  from  time  to  time, 
washed  with  water  and  examined  with  the  No.  7  objective. 
As  soon  as  the  bacilli  are  decolored  the  washing  in  alco- 
hol is  discontinued.  The  specimen  then  shows  bright  red 
spores  within  cells  that  are  almost  or  wholly  colorless. 
The  cover-glass  is  then  stained  for  a  short  time  with 
methylene  blue,  washed  with  water  and  examined.  The 
spores  should  be  stained  deep  red  while  the  bacillus  itself 
should  be  light* blue. 

Spores  may  be  readily  simple  stained  by  passing  the 
cover-glass,  after  it  has  been  fixed,  8  to  10  times  through 
the  flame.  Then  the  specimen  is  heated  for  1  to  2  min- 
utes with  carbolic-fuchsine. 

The  carbolic-fuchsine  solution  known  also  as  ZiehTs 
solution,  is  prepared  by  adding  1  g.  of  fuchsine  and  13  c.  c. 
of  absolute  alcohol  to  100  c.  c.  of  5%  carbolic  acid.  The 
solution  is  heated  on  the  water-bath  until  everything 
dissolves  and  the  solution  has  a  clear  bright  red  color. 


MEMORANDA. 


MEMORANDA. 


—  Ill  — 

SPORE  STAINS. 

Cover-glass  preparations. 
Air  dried. 


Simple: 

12  x  through  flame. 
Carbolic-fuchsine    (hot 

inin.). 

Water  (and  examine). 
Air  dried. 
Canada  balsam. 


Double: 

3  x  through  flame. 
Carbolic-fuchsine  (hot  2  to 

5  min  ). 

Water  (and  examine). 
Dilute  alcohol. 
Water  (and  examine). 
Contrast  color  (metbylene 

blue,  ]-  to  •£•  min.j. 
Water  (and  examine). 
Air- dried. 
Canada  balsam. 

2;  to  Metchnikoff's  cellular 
theory  of  immunity,  the  white  blood  cell  is  endowed  with 
the  power  of  taking  into  itself,  and  ultimately  destroying, 
the  invading  organism.  Phagocytic  action  can  be  readily 
demonstrated  in  frog-*  inoculated  with  anthrax.  For  this 
purpose  a  pure  culture  of  the  amiirax  baciilus  is  intro-. 
duced  into  the  dorsal  lymph  sac  of  a  frog,  and  at  the  end 
of  12  or  18  hours  it  is  killed  with  chloroform. 

Make  cover  glass  preparations  with  the.  fluid  in  the 
dorsal  lymph  sac  and  stain  some  with  simple  anilin  dyes 
and  others  after  Gram's  method. 


Pliagocytes.  —  Accordin; 


i  i 


—  112  — 

Summary  of  laboratory  work  with  anthrax: 
From  guinea-pig : 
Gelatine  plates, 

Colonies — Impression  preparations. 

Stich   cultures — Hanging-drop    and    permanent 

mounts. 

Agar  streak  cultures — Hanging-drop  and  perma- 
nent mounts. 
Ca   (OH)2-agar  streak  cultures — Hanging-drop 

and  permanent  mounts. 

Bouillon   hanging-drop    of  blood — threads  —  perma- 
nent mounts. 
Streak  preparations. 
Simple  stain. 
Gram's  stain. 

From  frog — phagocytes,  simple  and  Gram's  stain. 
Spores — simple  and  double  stain. 


MEMORANDA. 


—  114  — 

BACILLUS  ANTHRACIS. 

Davaine.     Pollender.     (1849). 

SYNONYMS  OF  ANTHRAX. — SPLENIC    FEVER   (IN  CATTLE)  :    WOOL-SORTER*  S 

DISEASE,  MALIGNANT  PUSTULE    (IN  MAN)  ;  MILZBRAND   (Germ.}  ; 

CHARBON.,   SANG  DE  KATE   (/*>.). 

Origin. — In  the  blood  and  tissues  in  anthrax. 

Form. — Large,  clear,  homogeneous  rods,  with  slightly  rounded 
ends ;  si/e  varies  witli  different  media,  but  the  length  is  less 
than  the  diameter  of  a  blood  cell.  Occurs  in  blood  in  short  threads 
of  2-4-0  cells,  which  may  show  slightly  swollen  ends.  In  bouillon 
and  on  agar  forms  long  threads.  Involution  forms. 

Motility. — lias  no  motion. 

Sporulation — Forms  median,  oval  spores,  without  enlarge- 
ment of  cell.  Alter  long  cultivation  it  may  lose  the  property  of 
forming  spores — asporogenic  variety.  I;  such  cases  the  addition  of 
fo-1  drop  of  Cu(OH)2  to  an  agar  tube  favors  spore  formation. 
Optimum  temperature,  30°  C.  Not  formed  below  18°  C.  Spores 
possess  variable  resistance.  Spores  not  formed  within  the  body. 

Anilin  Dyes. — Stu.n  readily,  also  by  Gram's  method. 

Growth. — Is  rapid. 

<;<'lii/iti  !'!«(cs. — Deep  colonies  form  round,  granular,  yellowish-brown 
masses,  with  irregular  borders.  Surface  colonies  are  very  characteristic,  and 
according  to  the  consistency  of  the  gelatin  the  border  is  fibril  I  tiled,  or  shows 
very  wavy  strands  of  threads— Medusa  head.  Liquefy. 

Mich  Cultures.—  .Short  threads  radiate  from  the  line  of  inoculation  into 
the  surrounding"  gelatin,  imparting  a  brush-like  appearance.  Cup-shaped 
liquefaction  forms  on  top  and  gradually  extends  till  the  contents  are  wholly 
liquefied.  The  mass  of  bacteria  settles  to  the  bottom  and  leaves  a  perfectly 
clear  solution  above,  without  scum. 

Streak  Cultures. — On  agar,  forms  a  dry,  grayish-white  growth  On 
potatoes,  the  growth  is  abundant,  white,  cream-like  and  rather  dry;  spores. 

Oxygen  requirements. — Is  aerobic,  but  can  grow  in  the  body 
as  a  facultative  amierobe. 

Temperature. — Grows  between  12  and  45°    C.     Optimum  :>7  . 

Behavior  to  Gelatin. — Liquefies. 

Attenuation — By  heating  for  ten  minutes  at  35°  C. ;  ^-1 
minute  at  100°.  By  growing  at  42.5°  for  four  weeks.  By  action  of 
chemicals,  mercuric  chloride,  carbolic  acid,  etc.  By  insolation.  By 
growth  under  pressure.  In  the  body  of  immune  animals,  as  frogs. 

Immunity. — Obtained  with  attenuated  cultures,  first  and 
second  vaccine  of  Pasteur;  with  sterilized  cultures;  with  extract  of 
thymus  gland  and  of  testes. 

Pathogenesis  — White  mice,  guinea-pigs,  rabbits,  sheep,  cat- 
tle, horses  and  man  are  susceptible.  Dogs,  old  white  rats,  birds 
and  frogs  are  insusceptible.  Subcutanec  us  application  kills  in  24- 
48  hours.  Post-mortem  shows  subcutaneous  oedema  and  enlarged 
spleen.  Bacilli  everywhere. 

Infection. — (1)  Through  the  food,  presence  of  spores, — Intes- 
tinal anthrax  in  sheep  and  '.attle.  (2)  Through  wounds, — Inocu- 
lation anthrax  in  man  (malignant  pustule),  (3)  Through  the  air, — 
Lung  anthrax  in  man,  the  wool-sorter's  disease  and  possibly  rag- 
picker's disease. 


MEMORANDA. 


—  116  — 

BACILLUS  OF  SYMPTOMATIC  ANTHRAX. 

Feser  and  Bellinger  (1878). 

SYNONYMS     OF    SYMPTOMATIC  ANTHRAX. — BLACK    LEG,     QUARTER    EVIL; 
CHARBON  SYMPTOMATIQUE   (Fr.)  ',  RAUSCHBRAND  (Germ.}. 

Origin. — In  the  subcutaneous  tissue,  muscles,  serous  exudate, 
etc.,  of  symptomatic  anthrax. 

Form. — Rather  large,  narrow  rods,  with  distinctly  rounded 
ends ;  almost  invariably  single,  may  form  in  twos.  About  three 
times  as  long  as  wide.  Involution  forms  appear  in  old  cultures — 
swollen  in  the  middle  or  at  the  ends. 

Motility.— Actively  motile.  Spore  bearing  rods  eventually 
lose  their  motion.  Shows  lateral  flagella,  also  giant  whips. 

Sporulation. — Spores  develop  readily  in  all  media  as  bright 
oval  bodies,  situated  near  one  end  which  is  somewhat  enlarged. 

Anilin  Dyes.— Stain  readily.  Not  by  Gram's  method.  Spores 
readily  double  stained. 

Growth. — Rapid,  and  gives  off  a  strong  butyric  acid  odor. 
Acid  or  alkaline. glucose  media  are  best.  Requires  anaerobic  con- 
tions. 

Plates. — On  gelatin,  forms  irregular  masses  surrounded  by  a  dense  whorl 
of  threads.  Liquefies.  On  ayar,  the  form  of  colonies  varies.  Usually  appears 
as  a  dense  mass  of  threads. 

Stick  Cultures.— In  glucose  gelatin  development  takes  place  in  the  lower 
part  of  the  tube;  the  contents  are  liquefied  and  gas  is  produced.  Energetic 
growth  and  gas  production  in  glucose  agar.  The  contents  of  the  tube  are  torn 
into  several  parts.  Giant  whips  common.  (NovY.) 

Streak  Cultures. — On  glucose  cigar,  in  hydrogen  forms  a  whitish  spread- 
ing film.  On  blood  serum  good  growths;  giant  whips  (Loftier). 

Bouillon.— Becomes  cloudy;  gas  bubbles  accumulate  on  the  surface; 
-after  several  days  the  growth  settles  to  the  bottom,  forming  a  compact, 
adherent  sediment.  Liquid  above  remains  cloudy  for  several  days. 

Glucose  gelatin,  colored  with  litmus,  develops  growth  in  incubator 
under  ordinary  conditions.  The  color  of  the  litmus  changes  to  a  wine-red, 
•showing  formation  of  acids.  Heavy  flocculent  sediment  on  the  bottom. 

Milk.— The  casein  is  coagulated.    Starch  is  not  inverted. 

Oxygen  requirements. — Is  an  obligative  anaerobe.  Grows  in 
vacuum,  hydrogen,  carbonic  acid,  etc. 

Temperature. — Grows  slowly  at  ordinary  temperature.  Best 
at  37-38°  C. 

Behavior  to  Gelatin.— Liquefies. 

Aerogenesis. — Energetic  production  of  gas,  having  a  disagree- 
able odor;  is  inflammable  and  consists  of  marsh  gas,  etc. 

Attenuation. — Bouillon  cultures  soon  lose  virulence  but  main- 
tain their  vitality.  Attenuation  takes  place  at  42-43°.  Spore  bear- 
ing material  heated  to  80°  and  100°  becomes  attenuated.  Virulence 
•restored  by  inoculating  animals,  and  at  same  time  injecting  some 
•lactic  acid.  Virulence  maintained  in  solid  media. 

Immunity. — Can  be  obtained  (1)  by  inoculating  small 
amounts  of  virulent  organism  ;  (2)  by  intravenous  injections;  (3) 
by  injecting  heated  cultures,  100°  and  80°  C. ;  (4)  with  inactive  old 
•cultures;  (5)  with  filtered  cultures. 

Pathogenesis. — Young  cattle,  sheep,  goats,  guinea-pigs  are 
highly  susceptible.  Horse,  ass,  white  rat  are  less  so;  while  hogs, 
dogs,  cats,  ordinary  rats,  rabbits,  doves,  ducks,  chickens  are  wholly 
immune.  Subcutaneous  injection  in  guinea-pigs  produces  death  in 
24-48  hours.  An  extensive  subcutaneous  oedema  is  present.  The 
muscles  are  dark  and  infiltrated. 

Infection. — Takes  place  naturally  by  inoculation  through 
wounds ;  not  through  the  food  or  air.  Poisoned  arrows  used  in 
fishing  in  Norway. 


MEMORANDA. 


—  118  — 

BACILLUS    CEDEMATIS    MALIGNI. 

Pasteur.    (1877). 

VIBRION  SEPTIQUE     OF    PASTEUR.       SYNONYMS    OF    MALIGNANT    (EDEMA. — 

SEPTICEMIE  (Fr.);  MALIGNES  ocDEM  (Germ.). 

Origin.— From  animals  inoculated  with  garden  soil;  from 
horse  and  from  man  (septicemie  gangretieuse). 

Form. — Rods  about  three  times  as  long  as  wide,  with  rounded 
ends;  usually  single,  but  may  form  threads,  especially  in  the  body. 
In  size,  etc.,  resembles  the  bacillus  of  S.  anthrax  ;  is  narrower  than 
anthrax  bacillus. 

Motility. — Actively  motile.  Show  lateral  flagella;  also  giant 
whips  (NovY). 

Sporulation. — In  bouillon  and  agar,  spores  appear  in  24  hrs. 
The  best  temperature  is  about  37°  C.  The  spores  are  median  or 
nearly  so,  with  corresponding  enlargement  of  the  parent  cell. 

Anilin  Dyes. — React  readily.  Is  stained  by  Gram's  method. 
Spores  stain  double. 

Growth  — Is  very  rapid,  especially  on  glucose  media.  Requires 
anaerobic  conditions. 

Plates.  —  On  gelatin,  colonies  develop  in  2-3  days,  and  under  the  micro- 
scope resemble  those  of  the  Hay  bacillus.  As  they  become  larger  gas  bubbles 
form.  On  agar  plates  at  37°  the  colonies  appear  as  an  irregular,  dense  net- 
work of  threads. 

&lich  Cultures. — In  gelatin,  growth  occurs  in  the  lower  part  of  the  tube; 
the  gelatin  is  liquefied,  gas  given  off  and  the  growth  settles  on  the  bottom. 
Agar  cultures  are  torn  into  several  parts  by  the  gas  which  is  formed.  In  the 
liquid  on  the  bottom  of  the  tube,  giant  whips  can  be  found  by  staining. 

Streak  Cultures.— On  agar,  offer  no  special  characteristics.  Grows  on 
potatoes  without  forming  a  scum. 

Bouillon.— Becomes  cloudy,  and  in  1-2  days  the  growth  settles  on  the 
bottom  as  a  low,  adherent  sediment,  and  in  a  few  days  the  liquid  becomes  clear. 

Glucose  gelatin,  colored  with  litmus.— In  air  at  37°  C.  is  liquefied  and 
litmus  first  reduced,  then  in  presence  of  oxygen  Becomes  red — acid  production. 

Milk.— Develops  a  good  growth;  a  part  of  the  casein  is  precipitated. 
Starch  is  not  changed  to  sugar. 

Oxygen  requirements.— Is  an  obligative  anaerobe.  Grows  in 
vacuum,  hydrogen,  carbonic  acid,  etc. 

Temperature. — Growth  is  best  at  the  temperature  of  the  body. 
Can  grow  at  ordinary  temperature. 

Behavior  to  Gelatin  — Liquefies. 

Aerogenesis. — On  glucose  media,  especially  when  distinctly 
alkaline,  it  gives  rise  to  the  production  of  gas. 

Attenuation. — Bouillon  cultures  retain  virulence  for  months. 

Immunity. — One  attack  of  malignant  (edema  does  not  protect 
against  a  second.  100  c.  c.  of  heated  or  filtered  cultures  injected 
into  guinea-pigs  in  three  portions  confers  immunity ;  6-8  c.  c.  of  the 
serous  exudate  accomplish  the  same  result. 

Pathogenesis, — Rabbit  susceptible — distinction  from  sympto- 
matic anthrax.  The  horse,  hog,  dog,  cat,  chicken,  dove,  guinea- 
pig  and  mice  are  susceptible.  Cattle  are  immune.  Subcutaneous 
inoculation  in  guinea-pigs  of  %  c-  c-  or  more  of  bouillon  culture 
produces  death  in  about  24  hours.  Marked  subcutaneous,  spread- 
ing, reddish  oedema.  Bacilli  present,  single  or  in  threads,  in  subcu- 
taneous tissue,  serous  surfaces  as  peritoneum,  etc. ;  scarce  in  the 
blood.  25-30  c.  c.  of  the  filtered  bouillon  culture,  injected  subcu- 
taneously,  kills  guinea-pigs. 

Infection. — Takes  place  exclusively  by  inoculation  through 
wounds.  Poisoned  arrows  of  the  New  Hebrides.  Rag-picker's 
disease. 


MEMORANDA, 


_120  — 

BACILLUS  CEDEMATIS  MALIGNI,  NO.  II. 

Novy.     (1893). 

Origin. — From  guinea-pigs  inoculated  with  milk  nuclein 
obtained  from  casein  by  digestion  with  artificial  gastric  juice. 

Form.— In  the  animal  body  it  occurs  usually  in  single  rods,  4-5 
times  as  long  as  wide ;  may  also'  occur  in  short  threads.  On  arti- 
ficial media  it  develops  as  straight  or  bent  rods,  sometimes  forming 
peculiarly  twisted  threads.  The  contents  are  often  granular,  and 
show  a  bright  body  at  one  end. 

Motility. — Possesses  a  slight  swaying  motion,  which  is  often 
absent.  Has  lateral  flagella,  and  in  pure  culture,  as  well  as  in  the 
animal,  it  gives  rise  to  giant  whips  which  may  attain  a  length  of 
40-50-72  microns. 

Speculation.—  Spore  formation  not  observed. 

Aniliii  Dyes. — Stain  readily.     Gram's  method  applicable. 

Growth. — Depends  upon  the  vitality  of  the  organism.  When 
taken  from  an  animal  it  grows  rapidly. 

Plates:— On  glucose  agar  good  colonies  develop  in  2-3  days  at  37°  C. 
Show  a  very  irregular,  fibril  ated  border,  and  often  give  rise  tongas  bubbles. 
May  contain  giant  whips. 

Stick  cultures.— Develop  only  in  the  lower  part  of  the  tube.  In  glucose 
agar  having  proper  alkalinity,  it  develops  rapidly,  forming  a  plainly  visible 
growth  along  the  line  of  inoculation;  the  agar  is  soon  torn  into  several  parts 
by  the  gas  that  is  produced.  Cultures  soon  die  out. 

Streak  cultures.— Develop  on  glucose  agar  only  when  oxygen  is  com- 
pletely excluded-.  It  forms  a  white  film  which  spreads  over  the  surface.  On 
acid  agar  involution  forms  develop. 

Bouillon.— An  excellent  growth  develops  which  in  24  hours  settles  to 
the  bottom  as  a  loose,  flocculent  sediment;  the  liquid  above  becomes  clear. 

Glucose  f/elatin,  colored  with  litmus. — Is  liquified  and  acid  is  produced — 
the  litmus  is  turned  red. 

Oxygen  requirements. — Is  an  obligative  anaerobe.  Grows  in 
vacuum,  hydrogen,  nitrogen,  carbonic  acid,  illuminating  gas. 

Temperature. — Does  not  grow  below  25°  C.  Optimum  temper- 
ature about  39°  C.  Can  withstand  freezing  for  24  hours. 

Behavior  to  Gelatin. — Liquefies. 

Aerogenesis. — In  alkaline  media  gives  rise  to  gases.  Volatile 
acids,  as  butyric  acids,  etc.,  are  formed  in  artificial  culture  and  also 
in  the  body  (of  rabbits). 

Attenuation. — Cultures  left  in  hydrogen,  or  exposed  to  light, 
lose  their  virulence.  Is  not  attenuated  when  left  in  the  dark  or 
when  frequently  passed  through  animals.  Lost  virulence  can  be 
reconstituted  by  inoculation  with  a  "mixed"  culture  containing 
Proteus  vulgaris. 

Immunity. — Not  conferred  by  a  non- fatal  inoculation,  or  by 
old,  weakened  cultures,  or  by  the  serous  exudate  of  the  pleural 
cavity. 

Pathogenesis. — Subcutaneous  injection  of  %  c.  c.  of  hydrogen 
bouillon  cultures  kills  guinea-pigs,  rabbits,  white  rats,  white  mice, 
doves,  in  12-24  hours.  Marked  subcutaneous  cedema  present; 
serous  exudates  in  thoracic  and  abdominal  cavities.  Cover-glass 
preparations  made  from  the  subcutaneous  tissue,  or  serous  surfaces, 
as  peritoneum,  shows  usually  enormous  numbers  of  bacilli,  and 
frequently  giant  wrhips  are  also  present. 


MEMORANDA. 


—  122  — 
BACILLUS  TETANI.     Nicolaier  (188-4J. 

SYNONYMS      OF      TETANUS. — LOCK     JAW  ;      \\TNDSTARRKRAMPF     (Germ.}. 
TETANOS — (Fr.). 

Origin. — Found  in  animals  that  diedof  tetanus  after  inoculation 
with  earth ;  from  traumatic  tetanus  of  man  and  animals ;  from 
head-tetanus. 

Form. — Large,  narrow  rods  with  rounded  ends ;  frequently 
forms  threads. 

Motility. — Is  motile.  Simple  stains  with  gentian  violet  of  old 
agar  culture  may  show  long  spirals. 

Sporulaticn. — Occurs  rapidly,  in  24-48hours  at  37°  C.  Forms 
terminal  spores,  with  enlargement— drum-sticks. 

Anilin  Dyes. — Stain  rapidly.  Gram's  method  is  applicable. 
Spores  can  be  double-stained. 

Growth  — Pure  cultures  obtained  by  heating  the  spore-bearing 
material  to  80°  C.,  to  destroy  the  ordinary  bacteria.  Growth  slow. 

Plates.— At  ordinary  temperature  colonies  develop  in  gelatin  in  4-7  days, 
and  resemble  those  of  the  Hay  bacillus  or  Proteus.  The  gelatin  is  slowly 
liquefied  and  gas  produced.  On  agar  plates  the  colonies  appear  as  fain't 
clouds  which,  under  the  microscope,  are  seen  to  be  made  up  of  a  whorl  of 
threads  which  are  finer  than  those  of  other  anaerobes. 

Stich  cultures.— Development  restricted  to  the  lower  part  of  the  tube. 
Cultures  of  glucose  gelatin  lubes  show  along  the  line  of  inoculation  a  cloudy 
growth,  radiating  outward  into  the  surrounding  gelatin;  resembles  that  of 
the  Root,  bacillus.  Eventually  the  gelatin  is  liquefied.  Gas  bubbles  present. 
In  glucose  agar  at  37°  C.  the  growth  is  sometimes  indistinct  and  shows  radi- 
ations. 

Streak  cultures.-  -On  glucose  (tf/nr  develop  rapidly. 

Houillnn.  At  :>7°  becomes  diffusely  cloudy  and  remains  so  for  several 
days;  eventually  the  growth  settles  to  the  bottom,  forming  a  scarcely  visible 
sediment— distinction  from  preceding  anaerobes. 

Glucose  geldfin  colored  with  litmus.— At  "7°  C.  becomes  liquefied;  a 
very  small  sediment  forms,  and  the  culture  remains  blue,  showing  absence  of 
acid  formation— distinction  from  preceding. 

Milk — Grows  well  in  milk  without  inducing  any  change.  Does  not 
invert  starch. 

Oxygen  requirements. — Is  an  obligative  anaerobe  Grows  in 
vacuum,  hydrogen,  nitrogen,  and  carbonic  acid. 

Temperature. — Does  not  grow  below  16°  C.  The  optimum  is 
about  38°  C. 

Behavior  to  Gelatin. — Liquefies. 

Aerogenesis. — Gives  rise  to  gaseous  products,  also  disagree- 
able penetrating  odor.  Hydrogen  sulphide. 

Attenuation. — Partial  loss  of  virulence  by  culture.  Thymus 
bouillon  attenuates. 

Immunity. — Iodine  trichloride;  thymus  bouillon  cultures; 
blood  serum  of  artificially  immunized  rabbits,  horse,  sheep,  dog ; 
milk  of  immunized  goat. 

Pathogenesis. — Man,  horse,  sheep,  guinea-pigs,  young  cattle, 
goats,  white  mice  and  white  rats,  are  susceptible.  Rabbits  and 
dogs  are  less  susceptible.  Ducks  and  chickens  are  immune.  The 
bacillus  is  present  at  the  point  of  inoculation,  although  in  small 
numbers.  Intensely  poisonous  products.  The  filtered  bouillon 
culture  in  a  dose  of  0.0002  c.  c.  kills  mice,  and  0.002  c.  c.  kills 
guinea-pigs. 

Infection. — Occurs  through  wounds,  Poisoned  arrows  of  the 
New  Hebrides. 


MEMORANDA. 


MEMORANDA. 


—  123  — 


CULTURES  OF  ANAEROBIC  BACTERIA. 

Obligative  anaerobic  bacteria,  those  which  grow  only 
in  the  absence  of  oxygen,  require  special  conditions  for  cul- 
tivation. Their  growth  is  favored  by  the  addition  of  1  to  2 
per  cent,  of  glucose  to  the  nutrient,  medium,  whether  gel- 
atin, bouillon  or  agar.  Freshly  prepared  media  are,  as  a 
rule,  best  adapted  for  culture  purposes. 

The  numerous  methods  which  have  been  proposed  for 
obtaining  growths  of  anaerobic  bacteria  can  be  classified 
under  the  following  heads: 

(1.)  Exclusion  of  oxygen. 

(2.)  Exhaustion  of  air. 

(3.)  Absorption  of  oxygen. 

(4.)  Displacement  of  air. 

(5.)  Cultures  apparently  in  the  presence  of  air. 

The  well-known  method  of  Liborius,  of  culture  in 
deep  layers  of  gelatin  or  agar,  depends  upon  the  exclusion 
of  air.  The  method  is  .simple  and  very  convenient.  The 
culture-tubes  contains  glucose  agar  or  gelatin,  l|-2  inches 
high.  Stich  cultures  are  made  in  the  usual  manner. 
Growth  develops  in  the  lower  two-thirds  of  the  medium, 
while  the  upper  layer  of  J  to  £  inch  serves  to  exclude  the 
air.  In  order  to  insure  complete  exclusion  of  oxygen,  the 
contents  of  an  ordinary  agar  orgelatin  tube  can  be  lique- 
fied and  then,  with  proper  precautions  against  contamina- 
tion, poured  on  top  of  the  inoculated  medium  and  quickly 
cooled.  This  extra  layer  is,  as  a  rule,  unnecessary. 

Colonies  of  anaerobic  bacteria  can  be  obtained  by 
making  ordinary  gelatin  or  agar  plates,  and  then  placing* a 
sterilized  glass  plate  on  top  to  exclude  oxygen. 

Vacuum  cultures  are  frequently  resorted  to.    Gruber's 


—  124  — 

tubes  with  constricted  necks  are  commonly  employed  for 
this  purpose.  The  air  is  pumped  out,  after  the  medium  is 
inoculated,  and  the  tube  is  then  sealed  in  a  llame. 

The  absorption  of  oxygen  can  be  accomplished  by 
means  of  an  alkaline  solution  of  pyrogallic  acid.  In 
Buclmer's  method  the  inoculated  tube  is  placed  within  a 
larger  tube,  which  is  closed  with  a  rubber  stopper,  and 
which  contains  on  the  bottom  the  pyrogallate  solution. 

The  displacement  of  air  by  some  inert  gas,  as  hydro- 
gen, is  frequently  made  use  of  in  cultivating  anaerobic 
bacleria.  Special  tubes,  as  those  of  Li  bonus,  have  been 
introduced  for  this  purpose.  A  current  of  hydrogen  is 
passed  through  the  inoculated  tube  until  all  the  air  has 
been  displaced,  after  which  it  is  sealed  in  a  ilame.  Plate 
cultures  in  hydrogen  can  be  readily  obtained  with  Botkin's 
apparatus  —  a  bell  jar  inverted  over  liquid  paraffin,  or 
mercury. 

Cultures  of  anaerobic  bacteria  can  be  readily  obtained 
by  inoculating  glucose  gelatin  tubes,  colored  with  litmus, 
and  placing  them  in  the  incubator.  Although  the  con- 
tents of  the  tubes  are  liquid,  and  apparently  the  air  has 
free  access,  yet  energetic  growth  takes  place.  These  cul- 
tures preserve  their  vitality  for  a  considerable  period  of 
time  and  have  the  additional  advantage  of  being  readily 
accessible. 

The  most  convenient  method  for  obtaining  cultures  in 
a  vacuum,  or  in  an  atmosphere  of  any  desirable  gas,  is  to 
use  some  form  of  bottle  in  which  the  ordinary  culture  tubes 
can  be  placed,  and  the  exhaustion  or  displacement  of  air 
carried  out.  Fig.  1  shows  such  a  bottle,  which  is  provided 
with  a  special  stopper  through  which  a  current  of  gas,  as 
hydrogen,  carbonic  acid,  etc.,  can  be  passed.  The  bottle  is 
sealed  air-tight  by  merely  turning  the  stopper  through 
ninety  degrees.  Fig.  2  shows  a  simple  and  eilicient  form 
of  bottle  in  which  the  same  result  is  obtained  by  means  of 
two  glass  stop-cocks. 

Ordinary  test-tubes  containing  glucose  gelatin,  bouil- 


Fig.  1. 


12 


Fig. 


MEMORANDA. 


—  125  — 

Ion,  agar,  etc.,  are  inoculated  in  the  usual  manner.  The 
projecting  part  of  the  cotton  plug  is  cut  off  close  to  the 
mouth  of  the  tube,  and  the  plug  slightly  raised,  with 
sterilized  forceps,  to  facilitate  diffusion  of  the  gas.  The 
tube  is  then  placed  in  the  bottle  by  means  of  a  pair  of 
long  forceps  and  the  apparatus  connected  with  a  Kipp's 
hydrogen  generator.  The  current  of  hydrogen  should  be 
passed  first  through  an  alkaline  solution  of  lead  acetate, 
and  then  through  a  six  per  cent,  solution  of  potassium 
permanganate.  After  passing  through  the  apparatus  the 
gas  passes  through  a  small  wash-bottle  containing  water, 
which  serves  as  a  valve.  If  carbonic  acid  is  used  it  should 
be  passed  through  a  saturated  solution  of  sodium  carbonate. 
A  rapid  current  of  gas  is  passed  through  the  bottle  for  1  to 
2  hours,  it  is  then  sealed  by  turning  the  stopper,  and  set 
aside  in  the  incubator  to  develop. 

The  apparatus  can  also  be  used  for  vacuum  cultures, 
in  which  case  it  is  connected  with  a  Chapman  aspirator 
and  the  air  pumped  out.  The  alkaline  pyrogallate  method 
can  be  employed  with  excellent  results. —  (NovY,  Central- 
Uatt  fur  Bakteriologie,  14,  581,  1893). 

By  far  the  easiest  and  simplest  method  for  obtaining 
plate  cultures  of  anaerobic  bacteria  is  that  devised  also  in 
this  laboratary.  The  apparatus,  which  has  the  form  of  a 
desiccator,  is  provided  with  the  special  stopper  seen  in 
the  bottle,  Fig.  1.  Petri  plates  are  placed  in  the  appara- 
tus, hydrogen  is  then  passed  through  for  1  to  2  hours, 
and  finally  it  is  sealed  by  turning  the  stopper. 

LABORATORY  WORK. — Liquefy  four  glucose  agar  tubes 
by  heating  in  the  water  bath  ;  then  allow  to  solidify  in  an 
upright  position.  When  cool  make  deep  stich  cultures  of 

Bacillus  of  symptomatic  anthrax. 
Bacillus  of  malignant  oedema. 
Bacillus  of  malignant  oadema,  No.  II. 
Bacillus  of  tetanus. 


—  126  — 

If  the  agar  in  the  tube  is  less  than  one  inch  high,  it 
will  be  necessary  to  pour  on  top,  after  inoculation,  the  con- 
tents of  another  agar  tube,  taking  care  to  sterilize  the 
mouths  of  both  tubes.  Set  aside  the  inoculated  tubes  in 
the  incubator  for  24  to  48  hours.  The  cultures  are  then 
examined  in  hanging-drop.  Simple  stains  are  made,  also 
double  stains  of  spores.  The  drop  or  two  of  liquid  which 
sometimes  accumulates  on  the  surface  of  the  agar,  and 
invariably  on  the  bottom  of  the  tubes  can  be  stained  for 
ordinary  nagella  and  for  giant-whips. 

Make  the  following  cultures  at  the  same  time  as  the 
preceding : 

(1)  Streak  culture  on  inclined  glycerine  agar  of  the 
tubercle  bacillus,  using  either  a  pure  culture,  or  a  tubercle 
from  a  guinea-pig  inoculated  with  tuberculosis.     Spread 
the  material  thoroughly  over  the  surface  of  the  medium. 

(2)  Streak  culture  on  ordinary  inclined  agar  of  the 
Achorion  Schonleinii — the  fungus  of  favus. 

(3)  Streak  culture  on  ordinary  inclined  agar  of  Actin- 
omyces — the  fungus  of  lumpy-jaw. 

After  these  three  inoculations  have  been  made  the 
cotton  plug  of  each  tube  is  cut  off  close  to  the  mouth  of 
the  tube,  and  this  is  then  sealed  either  with  a  rubber  cap, 
or  with  sealing  wax,  or  with  paraffin  of  a  high  melting 
point,  56°  C.  The  sealed  tubes  are  then  placed  in  the 
incubator  for  several  weeks. 

STAINING  OF  FLAGELLA. 

In  order  to  obtain  good  stains  of  nagella  special  care 
must  be  given  to  the  preparation  of  the  cover-glasses.  These 
must  contain  as  little  organic  matter  as  possible  in  order 
to  prevent  the  formation  of  a  dirty  precipitate  on  the 
cover-glass.  Excellent  cover-glasses  can  be  made  by 
dilution.  A  small  loopful  of  the  turbid  fluid  from  the 
bottom  of  the  agar  tube  culture  of  (Edema  bacillus  No.  II, 


MEMORANDA. 


MEMORANDA. 


—  127  — 

or  from  that  of  the  Bacillus  of  malignant  oedema,  is  trans- 
ferred to  a  large  drop  of  distilled  water  in  the  center  of  a 
wide  cover-glass.  By  means  of  a  straight  platinum  wire, 
three  transfers  are  made  from  this  drop  to  another  drop  of 
distilled  water  on  a  second  cover-glass.  This  second 
cover  glass  will  now  contain  only  a  small  number  of 
bacteria  and  very  little  foreign  matter.  By  means  of  a 
platinum  wire,  with  a  very  small  loop,  not  much  larger 
than  a  pin-head,  transfers  can  now  be  made  to  t>  or  8  clean 
wide  cover  glasses.  Each  small  loopful  is  spread  at  once 
over  as  much  of  the  surface  of  the  cover-glass  as  possible. 
The  thin  film  of  liquid  evaporates  almost  immediately, 
and  the  cover-glass  can  then  be  fixed  by  passing  it  once 
through  the  flame.  Overheating  the  cover-glass  is  very 
likely  to  destroy  the  slender  flagella. 

The  cover-glass,  with  the  specimen  side  up,  is  held  in 
a  pair  of  forceps  and  covered,  by  the  aid  of  a  pipette,  with 
Loffler's  mordant  solution.  The  cover-glass  is  then  held 
over  the  flame,  which  should  be  turned  low,  for  about  a 
minute.  The  liquid  should  be  warmed  so  as  to  give  off 
vapors,  but  should  not  be  actually  boiled.  As  fast  as 
evaporation  takes  place  fresh  mordant  solution  should  be 
added,  and  at  no  time  should  it  be  allowed  to  dry  down 
on  the  cover-glass. 

The  mordant  must  then  be  thoroughly  and  completely 
washed  off  the  cover-glass  by  a  jet  of  water.  If  the  edge 
has  dried  down,  it  should  be  loosened  with  a  pin  or  knife 
and  then  washed  off.  To  still  further  clean  the  cover- 
glass,  it  should  be  dipped  for  a  few  seconds  in  absolute 
alcohol  and  again  washed  with  water. 

The  mordanted  cover-glass  is  then  covered  with  a  satu- 
rated solution  of  anilin  water  fuchsine,  or  with  carbolic 
fuchsine,  and  heated  over  the  flame  for  1  to  2  minutes, 
observing  the  same  precaution  as  before.  The  specimen 
is  then  thoroughly  washed  with  water  and  examined  with 
the  -i1/  inch  oil-immersion  objective. 


—  128  — 

Summary  for  staining  flagella  : 

Dilution  cover-glass  preparation. 
Air-dried. 
1  x  through  flame. 
Mordant,  hot  (1  to  2  min.). 
Water. 

Alcohol  (few  seconds). 
Anilin-water  fuchsine,  hot  (1  to  2  min.). 
Water  (and  examine). 
Air-dried. 
Canada  balsam. 

The  mordant  employed  is  prepared  by  dissolving  20  g. 
of  tannic  acid  in  80  c.  c.  of  distilled  water.  To  10  c.  c.  of 
this  tannic  acid  solution,  add  5  c.  c.  of  ferrous  sulphate 
solution  (1-2),  and  1  c.  c.  of  saturated  alcoholic  solution  of 
fuchsine. 

The  stain  employed  is  made  by  adding  4  to  5  g.  of 
fuchsine  to  100  c.  c.  of  anilin  water  (p.  106).  Both  mor- 
dant and  stain  should  be  kept  warm  while  in  use. 


MEMORANDA. 


MEMORANDA. 


MEMORANDA. 


—  130  — 

EXAMINATION  OF  SPUTUM  FOR  THE  TUBERCLE  BACILLUS. 

Ziehl-Neelsen  Method. — A  ]oopful  of  the  sputum  is 
transferred  to  a  wide  cover-glass  and  thoroughly  spread 
over  the  surface.  It  is  then  allowed  to  dry  in  the  air,  or 
by  moving  it  to  and  fro  over  the  flame,  and  then  fixed  in 
the  usual  way.  The  cover-glass  is  held  in  the  forceps, 
specimen  side  up,  and  covered  with  carbolic  fuchsine  solu- 
tion. It  is  warmed  over  the  flame  for  1  to  2  minutes, 
avoiding  actual  ebullition,  and  then  washed  with  water. 
The  specimen  is  now  dipped  in  dilute  nitric  acid  (a  watch- 
glass  is  filled  with  water  and  3  or  4  drops  of  nitric  acid 
added),  for  a  few  seconds.  Then  transferred  to  dilute 
alcohol  (60  to  70X),  where  it  is  moved  about  till  it  is 
almost  decolored.  After  this  it  is  washed  with  water  and 
stained  for  a  few  seconds  with  methylene  blue.  The  lat- 
ter is  washed  off  with  water  and  the  specimen  examined 
under  the  microscope.  It  should  show  the  tubercle  bacil- 
lus stained  bright  red,  on  a  light  blue  back-ground. 

Heavily  stained  preparations  can  be  obtained  by 
floating  the  prepared  cover-glass  on  the  carbolic-fuchsine 
solution  for  15  or  30  minutes,  then  decoloring,  as  before. 

Cover-glass  preparation. 

Air-dried. 

3  x  through  flame. 

Carbolic-fuchsine,  hot,  (1  to  2  min.). 

Water. 

Dilute  nitric  acid  (few  seconds). 

Dilute  alcohol. 

Water. 

Methylene  blue  (i  to  %  min.). 

Water  (and  examine). 

Air-dried. 

Canada  balsam. 

A  2  per  cent,  aqueous  solution  of  anilin  hydrochloride 
can  be  used  to  excellent  advantage  instead  of  the  dilute 
nitric  acid. 


MEMORANDA. 


—  132  — 
BACILLUS  TUBERCULOSIS.     Koch.    (1882). 

TUBERCLE    BACILLUS. 

Origin. — In  tuberculosis  of  mammals.  Lupus  vulgaris.  The 
bacillus,  present  in  chicken  tuberculosis  is  distinct  from  that  in 
mammals. 

Form. — Very  narrow,  rather  long  rods  which  are  smaller  than 
the  diameter  of  a  red  blood  cell.  The  ends  are  distinctly  rounded 
and  the  bacillus  itself  may  be  straight  or  more  frequently  is  slightly 
bent  or  nicked.  Occurs  usually  single  but  may  form  short  threads 
of  3-6  cells.  In  the  sputum,  tissues,  etc.,  is"  frequently  found  in 
small  bunches. 

Motility. — Has  no  motion. 

Sporulation. — Frequently  shows  a  number  of  bright  bodies 
within  the  cell,  but  these  cannot  be  considered  as  true  spores. 
.The  bacillus  itself  possesses  a  relatively  high  power  of  resistance  to 
heat,  desiccation,  acids,  putref action,  etc. 

Anilm  Dyes  — Stains  very  slowly  and  difficultly  with  simple 
anilin  dyes;  readily  with  hot  carbolic-fuchsine,  or  anilin-water 
fuchsiue  or  gentian  violet.  When  once  stained  it  is  difficult  to 
decolor,  whereas  ordinary  bacteria  do  so  readily.  Specimens  from 
sputum  and  tissues  can  therefore  be  readily  double  stained — dis- 
tinction from  all  known  bacteria,  except  the  leprosy  bacillus.  Can 
be  stained  by  Gram's  method. 

Growth. — Takes  place  very  slowly,  requiring  usually  several 
weeks  to  become  clearly  visible.  Furthermore,  a  special  tempera- 
ture, at  or  near  that  of  the  body,  and  special  media  as  blood-serum 
or  glycerine-agar,  etc.,  are  necessary. 

Plntes — .No  growth  has  been  obtained  on  plates.  Colonies  can  be  readily 
obtained  by  making  successive  streaks  on  glycerine-agar  or  blood-serum. 
Colonies  obtained  direct  from  the  sputum  are  round,  white,  opaque,  and  raised, 
resembling  colonies  of  white  yeast.  On  subsequent  culture  the  colonies  are 
dry,  grayish  scales.  Under  the  microscope  they  appear  as  interwoven, 
twisted  strands  of  threads. 

Stick  Cultures.—  Can  be  obtained  on  glycerine-agav.  Growth  restricted 
to  the  upper  part  of  the  tube.  It  spreads  over  the  surface  as  a  thick,  raised 
plaque  which  at  first  is  white,  but  later  becomes  yellowish. 

Streak  Cultures.-— On  glycerine  agar  or  blood-serum  eventually  develops 
an  abundant  dry,  granular,  raised  growth,  which  at  first  is  grayish,  but  later 
takes  on  a  light  yellow  tinge.  Similar  growths  develop  on  potatoes. 

Jiouill on.— Grows  well,  especia'ly  on  the  surface  in  bouillon  which  con- 
tains the  usual  amount  of  glycerine,  5-6  per  cent.  Such  bouillon  cultures, 
filtered  and  concentrated,  constitute  the  so-called  tuberculin. 

Oxygen  requirements. — Free  access  of  oxygen  necessary  for 
growth.  Is  a  facultative  anaerobe  (FHAENKEL). 

Temperature. — The  optimum  is  about  37-39°C.  Slight  varia- 
tions above  or  below  this  stop  the  growth.  It  cannot,  therefore, 
grow  at  ordinary  temperatures. 

Behavior  to  Gelatin. — No  growth. 

Attenuation. — Slight  attenuation  probably  does  result  with 
age,  but  otherwise  it  has  not  been  positively  demonstrated. 

Pathogeneais. — Man,  monkeys,  cattle',  guinea-pigs,  field  mice, 
rabbits,  and  cats  are  susceptible.  White  mice,  rats,  and  dogs  are 
somewhat  insusceptible.  Chickens  are  immune.  Inoculation  of 
pure  cultures  produces  in  susceptible  animals  tuberculosis.  The 
formation  of  tubercles  and  of  giant  cells.  The  bacilli  may  be  very 
abundant,  at  other  times  are  scarce  and  difficult  to  find. 

Infection  — Takes  place  most  frequently  along  the  respiratory 
tract — Inhalation  tuberculosis.  May  occur  tlirough  wounds — Inocu- 
lation tuberculosis,  and  also  through  food — Intestinal  tuberculosis. 
Placental  infection. 


MEMORANDA. 


—  134  — 


BACILLUS  LEPRJE.     Hansen.     (1879). 

LEPROSY    BACILLUS. 

Origin.— Found  in  the  leprous  nodules  of  the  skin 
and  mucous  membrane,  lymphatic  glands,  liver,  spleen, 
marrow,  etc.  Not  in  the  blood. 

Form. — Rather  large,  narrow  rods,  which  resemble 
the  tubercle  bacillus. 

Motility .— Non-  motile. 

Sporulation. — Bright  bodies  frequently  observed 
within  the  cell,  as  is  the  case  of  the  tubercle  bacillus. 
Doubtful  if  these  are  spores. 

Anilin  Dyes.— Stain  readily.  Can  be  stained  by 
Gram's  method,  also  by  the  method  for  the  tubercle 
bacillus.  Sections  kept  in  alcohol  lose  the  property  of 
double  staining. 

Growth. — Has  not  been  obtained  under  artificial 
conditions  with  certainty.  The  cultures  of  Bordoni- 
UfFreduzzi  on  glycerine  blood-serum  inoculated  with  the 
marrow  of  long  bones.  Apparently  is  an  obligative  para- 
sitic organism. 

Pathogenesis.— While  the  constant  presence  of  the 
leprosy  bacillus  in  leprous  tissue  leads  to  the  prevailing 
view  that  it  is  the  cause  of  that  disease,  it  should  never- 
theless be  remembered  that  as  yet  unquestioned  pure 
cultures  have  not  been  obtained  and  hence  successful  in- 
oculations are  impossible.  Direct  infection  with  leprosy 
tissues  has  given  but  few  positive  results. 

Infection. — The  mode  in  which  this  occurs  in  man 
is  entirely  unknown. 


MEMORANDA. 


136  — 


AGAR  PLATE  CULTURES. 

The  ordinary  gelatin  plates  are  applicable  for  the 
isolation  of  colonies  of  only  those  organisms  which  can 
grow  at  ordinary  room  temperature.  Above  25°  C.  the 
nutrient  gelatin  melts  and  cannot  therefore  be  employed 
as  a  solid  medium  for  the  growth  of  those  organisms  which 
develop  only  at  a  higher  temperature.  In  such  cases  plates 
can  be  made  with  ordinary,  or  glycerine  or  glucose  agar. 
The  method  of  making  agar  plate  cultures  is  briefly  as 
follows: 

Three  agar  tubes  are  immersed  in  boiling  water  in  a 
water-bath  until  the  contents  are  liquefied.  The  burner  is 
then  removed  from  under  the  water-bath  and  the  water 
with  the  immersed  tubes  is  allowed  to  cool  slowly  until  a 
temperature  of  45°  C.  is  reached.  Tube  1.,  is  inoculated 
with  the  material  to  be  plated  and  the  usual  dilutions  to 
tubes  2  and  3  are  made  as  rapidly  as  possible.  The  cotton 
plugs  are  then  cut  off  short,  pushed  in  slightly,  and  the 
lips  of  the  tubes  sterilized  in  the  flame.  The  inoculated 
contents  are  then  poured  into  sterilized  Petri  dishes,  or  on 
ordinary  sterilized  plates.  Inasmuch  as  the  agar  solidifies 
at  about  40°  it  will  require  rapid  work  to  inoculate  the 
tubes  and  pour  the  contents  before  solidification  takes 
place.  Ice-water  must  not  be  used  to  congeal  the  agar. 
The  Petri  dishes  or  plates  are  then  placed  in  the  incubator. 

Esmarch  roll-tube  cultures  can  also  be  made  with 
agar  in  the  same  manner  as  described  for  gelatin  cultures. 
The  tubes  should  be  rotated  in  ordinary  tap  water. 

LABORATORY  WORK. — Make  glycerine  agnr  Petri 
dishes  from  the  spleen  of  a  guinea-pig  inoculated  with 
glanders.  When  the  colonies  develop,  make  streak  cul- 
tures on  inclined  glycerine  ngar.  Examine  the  spleen  and 
also  the  pure  cultures  in  the  usual  manner  by  making 
hanging-drops  and  cover-glass  preparations. 


MEMORANDA. 


—  138  — 
BACILLUS  MALLEI.    Loftier  and  Schtitz.  (1882). 

BACILLUS  OF  GLANDERS.    MORVE   (Fr.)  |  ROTZ  (Germ.}  }    MALLEUS  (.Ld£.). 

Origin. — Found  in  the  nodules,  ulcers,  discharges, 
etc.,  of  glanders  or  farcy. 

Form. — Rods  with  rounded  ends,  straight  or  slightly 
curved,  shorter  and  thicker  than  the  tubercle  bacillus. 
May  grow  in  pairs  or  in  short  threads. 

Motility. — Has  no  motion. 

Sporulatipn. — Bright  bodies  are  frequently  found 
in  the  cells,  as  in  the  tubercle  bacillus;  are  considered  by 
Loffler  as  the  first  indication  of  degeneration.  Keal  spores 
are  said  to  have  been  double  stained.  The  bacillus  itself 
is  highly  .resistant  to  desiccation. 

Anilin  Dyes. — Is  readily  stained  and  also  decolors 
rapidly.  Carbolic-fuchsine,  or  alkaline  anilin  gentian 
violet,  or  anilin  fuchsine  stain  well,  especially  when 
warmed.  Not  stained  by  Gram's  method. 

Growth. — Occurs  only  at  relatively  high  tempera- 
tures. Growth  is  rapid.  Glycerine  agaris  the  best  medium. 

Plates.— Cannot  be  obtained  with  gelatin.  On  glycerine  agar  at  37Q  C. 
forms  excellent  colonies  in  a  day  or  two.  These  are  round,  grayish,  and  glis- 
tening in  appearance,  with  granular  contents  and  smooth  sharp  borders. 

Stich  Cultures.—  Can  be  made  in  glycerine  agar,  not  in  gelatin. 

Streak  Cultures. --O\\  glycerine  agar  forms  a  thick,  moist,  slimy,  semi- 
transparent  growth.  On  potatoes  the  growth  is  very  characteristic.  At  first 
it  forms  a  thin,  transparent,  honey  or  amber-colored  growth  which  later  be- 
comes reddish-brown.  On  blood-serum  forms  yellowish,  transparent  spots 
which  eventually  fuse  together  and  yield  a  slimy,  whitish  growth. 

Bouillon.— Grows  readily  and  abundantly. 

Oxygen  requirements. — Is  a  facultative  anaerobe. 

Temperature. — Does  not  grow  below  25°  or  above 
42°  C.  The  optimum  is  about  37°  C. 

Behavior  to  Gelatin. — Scarcely  any  growth. 

Attenuation. — Takes  place  rapidly  when  grown  on 
artificial  media;  must  therefore  be  frequently  passed 
through  an  animal,  otherwise  the  virulence  is  lost  and  the 
organism  dies  out.  Mallein — the  filtered  cultures  of  the 
glanders  bacillus — analogous  to  tuberculin. 

Immunity.—  Sm all  amounts  of  bouillon  cultures  in- 
jected intravenously  into  dogs  confer  immunity. 

Pathogenesis. — Man,  horse,  ass,  guinea-pigs,  field 
mice,  cats,  and  goats  are  highly  susceptible.  Ordinary 
and  white  mice,  cattle,  and  hogs  are  immune,  while  dogs, 
rabbits,  and  sheep  are  slightly  susceptible.  White  mice 
become  susceptible  when  fed  with  phloridzin.  Susceptible 
animals  on  inoculation  develop  typical  glanders.  In 
guinea-pigs  death  results  in  4-6-8  weeks.  Field  mice  die 
in  a  few  days.  Enlarged  lymphatics,  nodules  in  liver, 
spleen,  etc.  Bacilli  present. 

Infection.— Through  wounds — inoculation  glanders. 
One  instance  in  man  with  pure  culture.  Along  the  re- 
spiratory tract — probably  the  usual  source  of  infection  in 
horses. 


MEMORANDA. 


—  140  — 
BACILLUS  DIPHTHEBIJE.  Klebs,  Loffler  (1883). 

BACILLUS    OF    DIPHTHERIA. 

Origin.— Found  in  diphtheritic  pseudo-membranes,  and  in 
very  small  numbers  in  the  spleen,  liver,  etc.,  of  diphtheria. 

Form. — Rather  large  thick  rods  which  are  straight  or  slightly 
bent  and  have  rounded  ends.  The  form  is  subject  to  considerable 
variation,  and  rods  with  swollen,  club-shaped  ends  are  frequently 
met  with — involution  forms. 

Motility.— Has  no  motion. 

Sporulation. — Spores  have  not  been  observed.  Tne  bacillus 
is  very  susceptible  to  desiccation,  or  to  heat  of  50°  and  above. 

Anilin  Dyes. — Dimple  anilin  dyes  react  poorly.  Can  be  best 
stained  with  carbolic-fuchsine,  or  with  Loffler's  alkaline  methylene 
blue  (30  c.  c.  of  cone,  alcoholic  solution  of  methylene  blue  +  100  c.c. 
of  a  0.01  per  cent,  solution  of  potassium  hydrate).  Is  also  stained 
by  Gram's  method. 

Growth. — Is  very  rapid  at  higher  temperatures  and  on  special 
media  as  glycerine  agar  and  blood  serum. 

Plates.— On  gelatin  plates  left  at  about  24°  C.  forms  very  small,  round, 
white  colonies  wh'ich  have  granular  contents  and  irregular  borders;  do  not 
liquefy  gelatin.  Cn  glycerine  agar  plates,  kept  in  the  incubator,  excellent 
colonies  form  in  24-48 'hours  The  deep  colonies  are  round,  or  oval,  coarsely 
granular.  The  surface  colonies  are  flat,  grayish  white,  glistening,  with  irregu- 
lar borders  and  coarsely  granular  contents. 

Rl.ich  Cultures.— In  gelatin  a  very  limited,  scarcely  preceptible  growth 
of  small,  round,  white  dots— marked  involution  forms  present. 

Streak  Cultures.— On  glycerine  agar  show  a  thin,  grayish,  spreading, 
adherent  film,  which  is  quite  characteristic.  On  potato  the  growth  is  invisible 
or  forms  a  dry,  thin  glaze — irregular  forms  of  the  bacillus  are  numerous.  On 
blood-serum  it  forms  a  thick  white,  opaque  growth. 

Bouillon.— Becomes  diffusely  clouded  and  the  growth  eventually  sub- 
sides on  the  sides  of  the  tube  and  on  the  bottom.  A  pellicle  may  form  on  the 
surface. 

Oxygen  requirements. — Is  a  facultative  anaerobe,  but  grows 
best  in  presence  of  oxygen. 

Temperature.— Very  slight  growth  at  20-25°  C.  The  maxi- 
mum is  about  42°  and  the  optimum  35-37°  C. 

Behavior  to  Gelatin. — Does  not  liquefy. 

Attenuation. — Cultures  directly  isolated  from  membranes 
show  frequently  marked  variation  in  virulence.  By  artificial  cul- 
ture the  virulence  is  still  further  diminished.  Cultures  can  be  at- 
tenuated by  growth  at  40°  in  a  current  of  air. 

Immunity. — Is  produced  by  filtered  bouillon  cultures  heated 
to  60-70°  C.  Also  by  injections  of  thymus  bouillon  cultures  prev- 
iously heated  to  65-70°.  Partial  results  with  iodine  trichloride. 

Pathogeiiesis. — Mice  and  rats  are  wholly  immune.  Finches, 
sparrows,  doves,  chickens,  rabbits,  guinea-pigs  and  cats  are  suscep- 
tible. Subcutaneous  inoculation  in  guinea-pigs  produces  death  in 
24-48  hours.  Pseudo-membranous  masses  form  at  point  of  inocula- 
tion ;  an  extensive  hemorrhagic  oedema  forms  under  the  skin  and 
exudates  occur  in  the  pleural  cavity.  Inoculation  in  the  trachea  of 
cats,  chickens,  doves,  rabbits,  etc.,  is  followed  by  pseudo-membrane 
formation,  and  by  death.  In  some  animals  as  rabbits  typical  diph- 
theritic paralysis  of  the  extremities  can  be  observed.  The  highly 
poisonous  toxalbumin. 

Infection. — Exact  mode  of  infection  is  not  known,  but  un- 
doubtedly occurs  through  the  air. 

NOTE.— Make  glycerine  agar  Petri  dishes  of  the  diphtheria  bacillus. 


MEMORANDA. 


—  142  — 

MICROCOCCUS  PNEUMONIA  CROUPOS.2E. 
Sternberg  (1880).    Frankel  (1883). 

SYNONYMS: — MICROBE   OF   SPUTUM   SEPTIC^MIA,   DIPLOCOCCUS  PNEU- 
MONIA, FKANKEL'S  DIPLOCOCCUS. 

Origin. — Occasionally  in  saliva  of  healthy  persons;  especially 
in  "  rusty  "  sputum  of  pneumonia.  The  same  organism  or  scarcely 
distinguishable  varieties,  are  present  in  cerebro-spinal  meningitis, 
pleuritis,  peritonitis,  pericarditis,  etc. 

Form. — Oval  or  lance-shaped  diplococci,  may  form  chains  of 
4-6  cells  and  resemble  a  streptococcus.  Owing  to  its  oval  form  it  is 
sometimes  regarded  as  a  bacillus.  In  the  animal  body  it  is  sur- 
rounded by  large  capsules. 

Motility. — Has  no  motion. 

Sporulation. — Unknown. 

Anilin  Dyes. — Stains  readily,  also  by  Grain's  method.  The 
•capsules  remain  colorless. 

Growth. — Takes  place  somewhat  slowly  and  only  at  higher 
temperatures,  and  on  alkaline  media. 

Plates. — On  gelatin  plates  kept  at  24°  C.  small,  round,  sharply  denned, 
tly  granular,  whitish  colonies  develop  slowly.     On  agar  plates  in  the  in- 
-cubator,  in  48  hours,  delicate,  glistening,  transparent  drops  form  which  under 


slightly  granular,  whitish  colonies  develop  slowly.     On  agar  plates  in  the  in- 
-cubator,  in  48  hours,  delicate,  glistening,  transparent  drops  form 
the  microscope  are  round,  sharply  bordered  and  finely  granular. 


Stich  Cultures.— In.  gelatin  a  row  of  small,  white  granules  develop  along 
the  line  of  inoculation.  Does  not  liquefy. 

Streak  Cultures.— On  agar,  in  the  incubator,  the  growth  develops  as  a 
thin  layer  of  delicate,  glistening,  almost  transparent  drops.  Dies  out  rapidly. 
On  blood-serum  forms  a  transparent  film-like  dew  drops.  No  growth  on 
potato. 

Bouillon. — Excellent  growth  occurs;  vitality  preserved  for  some  time. 

Milk. — Is  a  favorable  culture  medium,  becomes  coagulated. 

Oxygen  requirements.— Is  a  facultative  anaerobe. 

Temperature. — Growth  occurs  only  between  24  and  42°.  Its 
optimum  is  about  37°  C. 

Behavior  to  Gelatin, — Does  not  liquefy. 

Attenuation. — Cultures  from  different  sources  show  marked 
difference  in  virulence.  When  grown  on  artificial  media  it  rap- 
idly attenuates  and  soon  dies  out,  unless  it  is  passed  through  a  sus- 
ceptible animal,  as  a  rabbit,  every  few  weeks.  Rapidly  attenuates 
*it42°O. 

Immunity. — Intravenous  injection  of  very  small  amount  of 
virulent  culture ;  injections  of  filtered  cultures,  especially  when 
heated  to  00°  C. ;  blood  serum  of  immune  animals.  Blood-serum 
from  pneumonic  patients  immunizes  rabbits  against  the  pure  cul- 
ture. 

Pathogenesis. — Subcutaneous  injection  of  0.1 — 0.2  c.  c.  of 
bouillon  cultures  in  rabbits  produces  death  in  24-48  hours.  The 
•diplococcus  is  found  in  the  blood  and  internal  organs  and  is  sur- 
rounded by  a  capsule.  Tracheal  injections  in  rabbits  produce  true 
pneumonia.  Mice  and  rabbits  are  highly  susceptible  ;  guinea-pigs, 
sheep,  dogs  are  less  susceptible.  Is  the"  recognized  cause  of  croup- 
ous  pneumonia. 

NOTE.— Make  agar  Petri  dishes  from  the  peritoneum  exudate  in  a  rabbit. 
Make  cover-glass  preparations  from  the  peritoneum,  surface  of  intestines  and 
tieart-blood,  and  stain  by  simple  and  by  Gram's  method. 


MEMORANDA. 


—  144  — 

PNEUMOBACILLUS  OF  FEIEDLAENDER. 

(1883). 

ALSO  KNOWN  AS  FRIEDLAENDER's  PNEUMOCOCCUS. 

Origin. — Is  frequently  found  in  normal  saliva  ;  also 
in  lungs  and  "  rusty  "  sputum  of  pneumonia. 

Form. — May  appear  as  an  oval  coccus,  but  in  reality 
is  a  short,  thick  rod,  which  may  grow  in  pairs  and  even  in 

short  threads.     In  the  animal  body  it  is  enveloped  by  a 
capsule. 

Motility. — Has  no  motion. 

Sporulation. — No  spores  observed.  Cultures  retain 
vitality  for  many  months. 

Anilin  Dyes. — The  cell  is  stained  readily  but  the 
capsule  remains  colorless.  Is  not  stained  by  Gram's 
method — distinction  from  Rhinoscleroma  bacillus  and 
Frankel's  fiiplococcus. 

Growth.— Is  rapid  and  abundant. 

Platen. — On  gelatin  plales  it  develops  rapidly.  The  deep  colonies  are 
round  or  oval,  sharply  bordered,  finely  granular  and  yellowish.  The  surface 
colonies  are  quite  characteristic  and  appear  as  thick,  moist,  glistening,  white 
masses  which  do  not  lend  to  spread  but  rather  to  become  convex  and  raised. 
No  liquefaction. 

Stich  Cultures.- (Irmvth  takes  place  along  1he  entire  line  of  inoculation 
and  is  especially  developed  on  the  surface  forming  a  "  nail-shaped  "  culture. 
As  the  culture  becomes  old  the  gelatin  near  the  surface  becomes  brownish  in 
color  and  small  gas  bubbles  may  form. 

Streak  Cultures.— On  ac/ar  forms  a  thick,  white,  moist,  shiny  growth 
On  blood-serum  develops  as  a  grayish,  shinv  mass.  On  potatoes  forms  a  thick, 
yellowish,  sticky  growth,  showing  gas  bubbles. 

Oxygen  requirements.— Is  a  facultative  anaerobe. 

Temperature. — Grows  rapidly  at  low  temperatures, 
16-20;  also  in  the  incubator. 

Behavior  to  Gelatin.— Does  not  liquefy. 

Aerogenesis. — Abundant  production  of  gas  in  4  per 
cent,  gelatin  ;  potato  cultures  grown  in  the  incubator 
also  give  rise  to  gas. 

Pathogenesis. — Is  pathogenic  for  mice  and  young 
rats;  guinea-pigs  and  dogs  are  less  susceptible,  while  rab- 
bits are  immune.  While  not  the  cause  of  pneumonia,  its 
frequent  presence  in  that  disease  may  serve  to  bring  about 
a  "  mixed  infection." 

NOTE —Make  gelatin  plates  and  cover-glass  preparations  from  the 
lungs  and  blood  of  a  young  rat  which  has  received  an  intrapleural  injection. 


MEMORANDA. 


—  146  — 


BACILLUS  OF  RHINOSCLEROMA. 

Frisch  (1882). 

Origin. — In  the  tumors  of  rhinoscleroma,  a  rather 
rare  disease  occurring  in  Austria  and  Italy. 

Form. — Short,  thick  rods  with  rounded  ends  resem- 
bling the  Friedlaender's  pneumobacillus,  may  form  short 
threads.  Are  likewise  surrounded  by  a  colorless  capsule. 
Cells  of  Mickulicz. 

Motility.— Has  no  motion. 
Sporulation. — Not  observed. 

Anilin  Dyes. — Stain  readily,  and  show  colorless 
capsule;  also  stained  by  Gram's  method. 

Growth. — Growth  resembles  in  almost  every  respect 
that  of  the  Friedlaender  bacillus,  The  colonies,  stich  and 
streak  cultures,  agree  so  closely  as  to  be  scarcely  distin- 
guishable. 

Oxygen  requirements. — Is  a  facultative  anaerobe. 
Temperature. —Grows  rapidly  at  ordinary  tempera- 
ture.    The  optimum  is  about  36-38°  <J. 

Behavior  to  Gelatin.— Not  liquefied. 
Aerogenesis. — Gas  produced  by  potato  cultures. 

Pathogenesis. — The  relation  of  this  bacillus  to  rhin- 
oscleroma has  not  been  positively  established.  It  has  less 
virulence  than  the  Friedlaender  bacillus.  Is  pathogenic 
for  mice  and  guinea-pigs,  Rabbits  are  less  susceptible. 


MEMORANDA. 


—  148  — 
VIBRIO  OF  ASIATIC  CHOLERA.      Koch  (1884). 

BACILLUS,  COMMA    BACILLUS,  SPIRILLUM    OF    ASIATIC    CHOLERA. 

Origin.— In  the  excreta  of  cholera  patients,  also  in  the  intesti- 
nal contents  after  death.  Found  several  times  in  the  water  supply. 

Form. — Usually  appears  as  a  short,  rather  thick  rod  with 
rounded,  narrowed  ends,  and  with  a  more  or  less  decided  bend  or 
twist.  May  therefore  vary  from  apparently  a  straight  rod  to  one 
bent  in  the  form  of  a  half  circle.  Usually  the  bend  is  such  as  to 
resemble  a  comma,  hence  the  name,  comma  bacillus.  When  two 
cells  remain  attached  the  elongated  "  8  "  form  results.  Grown  in 
liquid  media  under  unfavorable  conditions  may  form  long  spirals. 
As  the  usual  form,  the  bent  rod,  is  a  segment  of  a  spirillum  it  is  des- 
ignated as  a  \  ibrio.  Peculiar  involution  forms  develop  in  old  cul- 
tures. 

Motility. — Is  actively  motile  ;  has  at  one  end  a  single  flageilum. 

Speculation. — Has  no  highly  resistant  form.  Has  been  said 
to  form  arthrospores.  • 

Anilin  Dyes. — Stains  slowly  with  simple  anilin  dyes.  Carbolic 
fuchsine  is  excellent.  Is  not  stained  by  Gram's  method. 

Growth  — Is  fairly  rapid  at  ordinary  temperature. 

Plates.— Un  gelatin  plates  kept  at  22°  C,  characteristic  colonies  develop 
in  15-20  hours.  The  colonies  appear  as  small,  white  points,  which  gradually 
reach  the  surface  and  produce  a  rather  slow  liquefaction  so  that  funnel-shaped 
depressions  form.  After  several  days  the  plate  becomes  wholly  liquefied. 
Under  the  microscope  the  colonies  show  an  irregular,  rough  border;  have  a 
white  or  pale-yellow  color,  and  the  contents  are  coarsely  granular,  as  if  made 
up  of  broken  glass.  A  faint  rosy  hue  surrounds  the  border.  On  agar  plates, 
at  37°  the  large  colonies  have  a  peculiar  bright  grayish-brown  transparent  ap- 
pearance, quite  distinct  from  common  bacteria  of  water  and  feces. 

Stich  Cu Mures.— Growth  in  gelatin  along  the  entire  line  of  inoculation. 
At  the  surface  a  funnel-shaped  liquelaction  forms  with  an  air  space  above, 
while  the  lower  part  contains  the  subsided  growth.  The  lower  part  of  the 
sticli  gradually  widens  by  liquefaction,  the  growth  settles  to  the  bottom  and 
eventually  the  entire  contents  of  the  tube  are  liquefied. 

Streak  Cultures.— On  agar  it  forms  a  bright,  glistening,  whitish  growth. 
Blood-serum  is  slowly  liquefied.  On  potatoes,  kept  in  the  incubator  it  forms 
a  grayish  or  yellowish  brown,  thin  uml  rather  transparent  lnyer,  which  re- 
sembles somewhat  that  of  glanders  bacillus  on  the  same  medium. 

Bouillon.— Growth  takes  place  rapidly,  especially  in  the  incubator,  and 
a  folded  scum  forms  on  the  surface.  Cultures,  12-^4  hours  old,  on  addition  of 
sulphuric  acid,  show  a  reddish  violet  color— the  indol  reaction— due  to  forma- 
tion of  indol  and  nitrous  acid. 

Milk.— Grows  abundantly  in  sterilized  milk,  without  much  change;  and 
also  in  sterilized  water. 

Oxygen  requirements. — In  artificial  cultures  requires  oxygen- 

Temperature. — Grows  oetween  35°  and  42°  C.  The  optimum 
is  at  or  about  37°  C.  Above  53°  it  is  killed. 

Behavior  to  Gelatin. — Liquefies. 

Pathogenesis. — Intravenous  injection  in  rabbits  kills  rapidly. 
In  guinea-pigs  intraduodenal  injections  or  introduction  of  cultures 
into  the  previously  alkalinized  stomach  produces  death  with  choleraic 
effects.  Intraperitoneal  injections  of  the  pure  bacillus  from  agar 
cultures  are  extremely  fatal  to  guinea-pigs,  producing  rapid  fall  of 
temperature.  Bacterial  cellular  proteids,  soluble  poisonous  pro- 
ducts. Infection  of  man  with  pure  cultures  produces  typical  cholera. 
It  is  therefore  recognized  and  accepted  as  the  cause  of  Asiatic 
cholera. 

Infection. — -Takes  place  along  the  alimentary  canal,  through 
the  water,  food,  contact  with  soiled  matter,  etc. 

NOTE.— Make  impression  preparations  of  the  colonies.  Also  place  in 
two  sterilized  plugged  tubes,  tap-water  solution  of  1  per  cent,  peptone  and 
1  per  cent.  Na  Cl.  Sterilize  the  peptone  tubes  in  steam  sterilizer.  Then  inocu- 
late with  cholera  vibrio,  place  in  incubator  over  night  and  then  apply  the 
indol  test. 


MEMORANDA. 


—  150  — 


VIBRIO  OF  FINKLEE,  AND  PRIOR.    (1884). 

VIBRIO    PROTEUS,  SPIRILLUM    OF    FINKLER   AND    PRIOR. 

Origin — In  the  dejections  of  cholera  nostras  which 
had  been  kept  fourteen  days.  Is  not  the  cause  of  cholera 
nostras. 

Form. — Resembles  in  form  the  cholera  vibrio  but 
is  larger  and  thicker;  occasionally  forms  short  spirals. 
Involution  forms. 

Motility. — Is  actively  motile  and  has  a  single  flagel- 
lum  at  one  end. 

Sporulation.— Has  not  been  observed. 

Anilin  Dyes.— Stains  readily  with  the  anilin  dyes. 

Growth. — Is  much  more  rapid  than  that  of  the 
cholera  vibrio. 

Plates. — The  colonies  develop  rapidly  on  gelatin  plates  and  produce 
extensive  circular  liquefactions  which  are  diffusely  clouded;  under  the  micro- 
scope they  appear  as  yellowish  brown,  finely  granular  masses,  the  contents  of 
which  can  be  seen  to  possess  marked  motion.  The  edge  is  beset  with  short 
delicate  fibrils. 

Stick  Culture*.— In  gelatin  tubes  rapid  growth  and  liquefaction  along  the 
entire  line  of  inoculation— the  so-called  stocking  shaped  liquefaction.  In  a 
few  days  the  entire  contents  are  liquefied  and  a  thin  film  usually  forms  011 
the  surface. 

Streak  Cultures. — On  agar  forms  a  thick,  moist,  slimy,  spreading  growth. 
On  potatoes  growth  occurs  rapidly  even  at  ordinary  temperature  forming  a 
grayish  yellow,  slimy,  glistening,  spreading  mass.  On  blood  serum,  rapid  de- 
velopment with  liquefaction. 

Milk.  —  Can  grow  in  milk,  but  in  water  it  soon  dies  out.  Abundant- 
growth  in  bouillon. 

Oxygen  requirements.— Is  a  facultative  anaerobe. 

Temperature. — Grows  well  at  ordinary  temperature, 
also  in  the  incubator. 

Behavior  to  Gelatin.— Liquefies  rapidly  and  exten- 
sively. 

Aerogenesis.— Not  observed. 

Pathogenesis. — As  a  rule  it  is  fatal  to  guinea-pigs 
when  bouillon  cultures  are  introduced  into  the  previously 
alkali nized  stomach.  The  intestines  are  pale  and  contain 
watery  contents. 


MEMORANDA. 


—  152  — 


VIBRIO  OF  DENEKE.     (1885). 

SPIRILLUM    TYKOGENUM.       SPIRILLUM    OF    DENEKE.       CHEESE    SPIRILLUM. 

Origin.— From  old  cheese. 

Form—  Sliirhtly  bent  rods,  which  forms  spirals,  and 
resemb'e  greatly  the  cholera  vibrio. 

Motility.— Very  motile. 

Speculation.— Not  known. 

Anilin  Dyes.— Stain  readily. 

Growth.— Is  quite  rapid,  but  less  than  (hat  of  the 
Finkler-Prior  vibrio.  On  some  gelatin  media  it  liquefies 
very  slowly  or  not  at  all. 

Plates. — The  colonies  which  develop  on  c/clatin  plates  are  yellowish, 
liquefy  and  the  plate  as  a  whole  may  resemble  somewhat  that  of  the  cholera 
vibrio,  but  the  liquefaction  is  more  rapid.  Under  the  microscope  they  are 
seen  to  be  irregular  coarsely  granular,  and  the  center  is  yellowish-green. 

8ticJi  Cultures.—  Growth  and  liquefaction  takes  place  in  gelatin  tubes 
along  the  entire  line  of  inoculation.  The  mass  of  bacteria  settle  to  the  bottom 
while  on  the  surface  a  yellowish  scum  or  layer  forms. 

Streak  Cultures.— On  agar  a  thin  yellowish  growth  develops  along  the 
line  of  inoculation.  On  potatoes,  in  the  incubator,  it  forms  a  delicate,  yel- 
lowish covering  which  frequently  contains  well-formed  spirals.  On  blood 
serum,  cultures  soon  produce  liquefaction. 

Oxygen  requirements.— Is  a  facultative  anaerobe. 

Temperature. — Grows  at  ordinary  temperature,  also 
at  37°  0. 

Behavior  to  Gelatin. — Liquefies  more  rapidly  than 
the  cholera  vibrio. 

Aerogenesis.— Not  observed. 

Pathogenesis — Is  less  pathogenic  to  guinea  pigs 
lhan  the  Finkler  Prior  vibrio. 


MEMORANDA. 


'  R  y 
THE 


V 

M 


OF 


—  154  — 


VIBRO  METCHNIKOVI.     Gamaleia  (188S.) 

SPIRILLUM    OP    METCHNIKOFF. 

Origin.— From  the  intestinal  contents,  blood  and 
organs  of  chickens  afflicted  with  a  disease  resembling 
chicken  cholera.  The  disease  exists  in  Russia  during  the 
summer  months  and  is  due  to  this  organism. 

Form.— Occurs  as  a  bent  rod  which  bears  a  marked 
resemblance  to  the  vibrio  of  Asiatic  cholera,  although  it 
is  somewhat  shorter  and  thicker  and  more  decidedly  bent. 
Has  a  marked  tendency  to  form  spirals.  In  the  animal 
body  it  is  very  short. 

Motility.— Actively  motile.  Possesses  a  long,  slen- 
der wnip  at  one  end. 

Sporulation.— Has  not  been  positively  shown  to 
have  spores.  Is  readily  destroyed  like  the  cholera  vibrio 
by  heat,  desiccation,  acids,  etc. 

Anilin  Dyes.— Stains  fairly  well— bi-polar  stain; 
not  by  Gram's  method. 

Growth.— In  the  hanging-drop  and  in  stained  prepa- 
rations this  organism  can  scarcely  be  distinguished  from 
the  cholera  vibrio.  The  cultural  properties  show  some 
diiferences,  and  especially  is  this  seen  in  the  pathogenic 
action  on  animals.  The  rapidity  of  growth  is  greater 
than  that  of  the  cholera  vibrio  and  less  than  that  of  the 
Finkler- Prior  vibrio. 

Plates.— The  colonies  on  gelatin  plates  may  resemble  those  of  the  cholera 
vibrio  and  also  those  of  the  Finkler-Prior.  As  a  rule  it  grows  and  liquefies 
more  rapidly  than  the  cholera  vibrio. 

Stich  Cultures.— In  gelatin  tubes  the  growth  resembles  exactly  that  of 
the  cholera  vibrio,  which  is  about  twice  as  old. 

Streak  Cultures. — On  agar  the  growth  resembles  that  of  the  cholera 
vibrio.  On  potatoes  in  the  incubator  it  develops  moderately  as  a  yellowish 
brown  covering. 

Bouillon.— Abundant  growth  in  the  incubator.  Gives  the  indol  reaction 
almost  as  well  as  the  comma  bacillus. 

Oxygen  requirements. — Same  as  cholera  vibrio. 

Temperature. — Same  as  cholera  vibrio. 

Behavior  to  Gelatin.— Grows  and  liquefies  more 
rapidly. 

Immunity. — Is  conferred  on  pigeons  and  guinea-pigs 
by  injection  of  sterilized  cultures. 

Pathogenesis. — Is  very  infectious  for  guinea-pigs, 
pigeons,  and  chickens;  rabbits  are  also  susceptible.  Pigeons 
die  after  subcutaneous  injection  of  minute  amounts  with- 
in 2-i  hours.  The  vibrios  are  abundant  in  the  blood  and 
internal  organs.  Toxicity  of  old  sterilized  cultures.  Rapid 
fall  in  temperature. 


MEMORANDA. 


—  156 


BACILLUS  NEAPOLITANUS.    Emmerich. 

(1884). 
EMMERICH'S  BACILLUS. 

Origin — Found  first  in  the  blood,  organs  and  intestinal 
contents  of  cholera  cadavers  in  Naples.  Has  since  been 
shown  to  be  normally  present  in  feces  ;  it  occurs  in  air  and 
in  putrid  fluids.  Is  closely  related  to  the  B.  coli  comraunis. 

Form. — Small,  short  rods  with  rounded  ends ;  usually 
single,  rarely  in  long  threads. 

Motility. — Has  no  motion.  Brownian  movement 
marked. 

Sporulation. — Not  observed.  When  desiccated  re- 
tains it  vitality  for  some  time. 

Anilin  Dyes.— Stains  readily,  but  not  by  Gram's 
method. 

Growth. — Is  rather  rapid,  even  at  ordinary  temper- 
ature. 

Plates.— On  gelatin  plates  the  deep  colonies  are  small,  round  or  whet- 
stone shaped,  yellowish-brown,  finely  granular  and  may  show  concentric 
rings.  The  surface  colonies  spread  freely  as  a  thin  plaque  with  irregular  or 
wavy  border.  A  delicate  marking  or  venation  can  be  seen  in  young  colonies. 
No  liquefaction. 

Stick  Cultures.— Are  characterized  by  a  pronounced  surface  growth  on 
the  gelatin.  It  spreads  rapidly  over  the  surface  as  dry,  grayish  white,  irreg- 
ularly bordered  mass.  Abundant  growth  along  the  stich.  In  old  cultures  the 
gelatin  frequently  becomes  cloudy — due  to  acid  production — and  bundles  of 
crystals  appear. 

Streak  Cultures.— On  agar  forms  a  moist,  white,  spreading  growth.  On 
potatoes  it  develops  as  a  yellowish- brown  sticky  mass. 

Oxygen  requirements. — Is  a  facultative  anaerobe. 

Temperature. — Grows  readily  at  ordinary  tempera- 
ture and  at  37°  C. 

Behavior  to  Gelatin. — Does  not  liquefy. 

Aerogenesis. — Not  observed. 

Pathogenesis. — Fatal  results  in  guinea-pigs,  rabbits, 
etc.,  may  follow  the  injection  of  large  quantities  of  the 
pure  culture.  It  is  a  toxicogenic  organism. 


MEMORANDA. 


—  158  — 


BACILLUS  COLI  COMMUNIS.    Escherich  (1885). 

BACTERIUM  COLI  COMMUNE  ',    THE  COLON  BACILLUS  OF  ESCHERICH. 

Origin. — Is  very  common  and  constant  in  the  intes- 
tinal contents  of  man  and  animals,  especially  in  the  colon; 
•occurs  in  the  discharges  of  healthy  infants,  also  in  summer 
diarrhoea.  Is  frequently  present,  accompanying  the  com- 
ma bacillus,  in  the  discharges  of  Asiatic  cholera,  and  in 
later  stages  may  be  the  only  organism  present.  In  many 
respects  it  resembles  Emmerich's  bacillus,  and  also  the 
typhoid  fever  bacillus. 

Form. — Short,  narrow  rods ;  may  vary  in  length  from 
oval  or  coccus-like  forms  to  rods  4-6  times  as  long  as  wide. 
Usually  grows  in  pairs,  arranged  in  groups. 

Motility. — Very  slowly  motile. 

Sporulation.— Not  observed. 

AnilinDyes. — Stains  readily;  not  by  Gram's  method. 

Growth. — Is  fairly  rapid  and  the  cultural  properties 
lesemble  those  of  the  preceding  organism. 

Plates.— On  gelatin  plates  the  surface  colonies  are  flat,  spreading,  an- 
isodiametric  and  have  a  dull-white  color.  The  border  is  irregular  and  mark- 
ings are  present  in  the  outer  zone.  No  liquefaction. 

Stich  Cultures.— Rather  energetic  growth  along  the  line  of  inoculation, 
while  on  the  surface  it  spreads  as  a  delicate  white  film. 

Streak  Cultures.— On  agar,  are  moist,  white,  spreading,  as  with  the 
Emmerich  bacillus.  On  potatoes,  it  forms  an  abundant,  yellowish,  moist, 
slowly  spreading  growth. 

Milk.—Ls  coagulated  in  7- 10  days. 

Oxygen  requirements. — Is  a  facultative  anaerobe. 

Temperature. — Grows  well  at  ordinary  temperature. 
Its  optimum  is  about  37°  0. 

Behavior  to  Gelatin. — Does  not  liquefy. 

Aerogenesis. — Under  anaerobic  conditions  produces 
•carbonic  acid  and  hydrogen. 

Pathogenesis. — Guinea-pigs  are  very  susceptible; 
rabbits  less  so;  mice  insusceptible.  Small  quantities  in- 
jected subcutaneously  or  intravenously  produce  diarrhoea, 
collapse  and  death  in  1-3  days.  The  small  intestine  is 
hyperaemic,  more  or  less  intensely  inflamed  ;  serous  exu- 
viates may  be  present.  The  bacilli  are  abundant  in  the 
blood  and  organs. 


MEMORANDA. 


—  160  — 


BACILLUS   TYPHI  ABDOMINALIS. 

Eberth(lS80). 

BACILLUS  OF  TYPHOID  FEVER J    KOCII-EBERTIl's    BACILLUF. 

Origin. — First  obtained  from  the  spleen  and  lymphatic  glands 
of  typhoid  fever  cadavers;  present  in  the  blood,  in 'small  numbers, 
and  in  the  feces  of  typhoid  patients. 

Form. — Well  formed  rods,  3-5  times  as  long  as  wide,  with 
rounded  ends.  The  length  varies  greatly  with  the  nature  of  the 
medium  on  which  it  grows.  Thus  on  agar  it  forms  very  short  rods, 
while  on  potatoes  it  may  grow  into  long  threads.  Involution  forms. 

Motility. — Is  very  actively  motile.  Has  numerous  lateral 
whips. 

Sporulation. — Terminal,  round  or  oval  bodies  are  found  in 
potato  and  agar  cultures  which  are  grown  in  the  incubator  for  sev- 
eral days.  They  do  not  double  stain  and  the  bacilli  containing 
these  are  very  susceptible  to  heat.  They  are  not  therefore  true 
spores.  The  bacilli  are  very  resistant  to  desiccation  and  may  retain 
vitality  for  months. 

Anilin  Dyes. — Does  not  stain  as  well  with  ordinary  anilin 
dyes  as  most  bacteria;  carbolic-fuchsine  stains  excellently.  Does 
not  stain  by  Gram's  method. 

Growth. — Is  rather  rapid,  In  its  cultural  properties  it  greatly 
resembles  the  two  preceding  organisms. 

Ptdfcx.  —  The  deep  co'onies  on  gelatin  plates  are  small,  round,  sharply 
bordered,  finely  granular  and  yellowish.  Th-  surface  colonies  spread  freely 
as  an  almost  transparent  film,  which  has  an  irregular,  wavy  horder,  and  is 
delicately  marked  with  branching  lines.  No  liquefaction. 

Stich<  ullun'x  —Abundant  growth  along  tn<>  entire  line  of  inoculation, 
and  especially  so  on  i  he  surface  wh^re  it  spreads  as  a  thin,  grayish  white 
covering.  Gelatin  eventually  becomes  cloudy,  due  to  the  production  of  acids. 

Streak  Cnttui'm. — Jn  ut/nr  and  on  hi •>')<!  serutn,  forms  moist,  white 
growth,  without  any  special  characteristics.  On  potatoes,  as  a  rule,  the  growth 
is  very  characteristic.  It  covers  the  surface  as  a  moist,  invisible  layer— -dis- 
tinction from  preceding  organisms.  On  alkaline  potatoes  the  growth  is  yel- 
lowish and  no  longer  characteristic. 

Oxygen  requirements. — Is  a  facultative  anaerobe. 

Temperature  — Grows  well  at  ordinary  temperature.  The 
optimum  is  about  37°  C.  Is  quicklv  killed  by  even  short  exposures 
to  heat  of  60°. 

Behavior  to  Gelatin. — Does  not  liquefy. 

Pathogenesis. — Intravenous  injections  usually  kill  rabbits. 
Is  usually  fatal  to  guinea-pigs,  when  introduced  into  the  previously 
alkalinized  stomach,  or  when  injected  into  the  duodenum.  Intoxi- 
cation. This  bacillus  is  generally  accepted  as  the  cause  of  typhoid 
fever. 

Infection. — Commonly  takes  place  through  the  mouth  by 
means  of  water,  food,  contact  with  soiled  articles,  etc.  In  later 
stages  other  organisms  as  streptococci  frequently  appear — "mixed 
infection." 

NOTE.— Make  Esmarch  potato  cultures  of  the  Eberth,  coli  communis, 
and  Emmerich  bacilli;  place  in  the  incubator  for  2-8  days.  Carefully  compare 
these  as  well  as  the  cultures  on  other  media,  etc.  Make  impression  prepara- 
tions of  colonies. 


MEMORANDA. 


—  162  — 


STREPTOCOCCUS  ERYSIPELATIS. 

Fehleisen  (1883). 

Origin. — Occurs  in  erysipelas,  in  the  lymphatic 
vessels  of  the  diseased  skin;  very  rarely  present  in  the 
blood  and  internal  organs. 

Form. — Small,  spherical  cells,  which  (end  to  grow  in 
chains  of  6-8  ;  not  infrequently,  as  when  grown  in  bouillon, 
the  chains  may  consist  of  a  hundred  or  more  cells. 

Motility. — Has  no  motion. 

Sporulation.  —  None.  Arthrospore  formation  has 
been  suggested. 

Anilin  Dyes. —Stain  readily;  Gram's  method  is 
applicable,  and  this  is  true  of  most  micrococci. 

Growth. — Is  readily  obtained  on  various  media,  even 
at  ordinary  temperature,  but  the  growth  is  slow  and 
limited. 

Plates.  -  On  gelatin  plates,  the  colonies  develop  rather  slowly,  forming 
minute,  round,  yellowish-brown,  finely  granular  colonies,  which  are  sharply 
bordered  and  usually  show  concentric  rings.  No  liquefaction.  On  affar 
plates,  developed  in  the  incubator,  it  forms  delicate,  grayish,  translucent, 
drop-like  colonies. 

8lich  Vultures. — In  gelatin  the  growth  is  quite  characteristic.  Along  the 
line  of  inoculation  a  row  of  minute  colonies  forms,  which  usually  remain  sep- 
arate, but  may  fuse  together,  giving  rise  to  a  continuous  stich.  Scarcely  any 
growth  forms  on  the  surface. 

Streak  Cultures.— On  agar  or  blood-serum,  it  develops  as  minute,  scarcely 
visible,  round  colonies,  which  do  not  tend  to  spread.  Growth  oil  potato  is 
doubtful. 

Bouillon.— At  37°  C.  soon  becomes  diffusely  clouded  and  a  slight,  whitish 
sediment  forms. 

Oxygen  requirements.— Is  a  facultative  anaerobe. 

Temperature. — Grows  slowly  at  ordinary  room  tem- 
perature ;  best  at  30-37°  0. 

Behavior  to  Gelatin. — Does  not  liquefy. 

Attenuation. — The  virulence  of  the  organism  is  sub- 
ject to  considerable  variation,  even  when  taken  directly 
from  a  case  of  the  disease.  Artificial  cultures  soon  become 
attenuated. 

Pathogenesis.— Man,  rabbits  are  susceptible.  Mice 
are  wholly  immune.  Typical  erysipelas  results  from  in- 
oculation with  pure  cultures.  Inoculations  in  man  in 
capes  of  inoperable  carcinoma,  etc.  Infection  in  young 
rabbits  frequently  gives  rise  to  suppuration,  severe  general 
symptoms,  elevation  of  temperature,  and  death. 

Infection. — Occurs  undoubtedly  through  wounds  or 
injuries  of  the  skin. 


MEMORANDA. 


—  164  — 


STREPTOCOCCUS  PYOGENES. 

Rosenbach  (1884). 

Origin. — In  abscesses,  pyaemia,  etc.  Similar,  if  not 
identical  streptococci,  are  to  be  met  with  under  most 
varied  conditions.  Tims  I  hey  are  found  in  the  mouth  and 
sputum,  on  the  mucous  membranes  of  the  nose,  urethra, 
vagina,  etc.  They  are  also  frequently  present  as  a  result 
of  •' mixed  in  e*-!ion  '*  in  diphtheria,  typhoid  fever,  pneu- 
monia, tuberculous-,  scarlet  fever,  etc. 

Form. — Spherical  cells  or  cocci  which  may  grow  in 
pairs,  but  usually  forms  rosary-like  chains  of  20  to  SOcells. 
Almost  identical  with  the  streptococcus  of  erysipelas. 

Motility.— None. 
Sporulation. — None. 

Anilin  Dyes.— Is  stained  readily;  also  by  Gram's 
method. 

Growth. — The  characteristics  of  growth  on  artificial 
media  are  essentially  the  same  as  those  of  the  erysipelas 
sireptococcus.  So  much  so  that  the  two  organisms  are 
frequently  considered  as  one  and  the  same  species. 

Oxygen  requirements. — Is  a  facultative  am,e;obe. 

Temperature.— Grows  slowly  at  room  temperature; 
best  at  35-37°  0. 

Behavior  to  Gelatin.— Does  not  liquefy. 

Pathogenesis. — Mice  and  rabbits  are  susceptible — 
distinction  Irom  the  sireptococcus  of  erysipelas.  Like  the 
Fiankel  diplococcus,  it  is  a  widely  distributed  engenderer 
of  inflammatory  processes,  either  in  connection  with  other 
diseases  as  diphtheria,  scarlet  fever,  etc.,  or  by  itself  pro- 
ducing effects  depending  largely  upoh  the  locus  of  inocu- 
lation. Thus  it  is  apparently  the  sole  cause  of  puerperal 
fever;  infection  of  the  lymphatics  of  ihe  skin  induces 
erysipelas,  while  subcutaneously  purulent  erysipelatoid 
changes  results.  Is  also  considered  the  cause  of  endo- 
carditis although  this  at  times  may  be  due  to  other  organ- 
isms which  have  the  similar  property  of  inducing  inflam- 
matory changes,  as  the  Fiankel  diplococcus  and  theStaphy- 
lococcus  pyogenes  aureus. 


MEMORANDA. 


—  166  — 

STAPHYLOCOCCUS  PYOGENES   AUREUS. 
Rosenbach  (1884). 

GOLDEN    PUS    PRODUCING    COCCUS. 

Origin. — One  of  the  most  common  organisms  in  pus 
—in  about  80  per  cent.  Has  been  found  on  the  surface  of 
the  skin,  in  saliva,  air,  water,  dust  and  soil. 

Form. — Smail  cocci,  arranged  in  irregular  groups; 
may  also  grow  single  or  lorm  diplococoi.  Size  varies  with 
the  medium. 

Motility. — Has  no  motion. 

Sporulation. — No  spores  observed.  Possesses  a  high 
degree  of  resistance  to  desiccation,  heat,  chemicals,  etc. 

Anilin  Dyes.  —  Siains  readily,  also  by  Gram's 
method. 

Growth.— Is  rapid. 

Plates.— On  (jelatin  plates  the  colonies  are  round,  with  sharp  smooth 
borders,  strongly  granular  and  of  a  dark-brown  or  yellow  color.  The  gelatin 
is  liquefied  somewhat  rapidly.  On  cigar  the  surface  colonies  are  bright  yellow 
in  color. 

Slich  Cultures.— In  gelatin  development  takes  places  along  the  entire 
line  ol  inoculation,  forming  a  finger-shaped  liquefaction.  The  growth  settles 
to  the  bottom  as  a  yellowish  deposit  while  the  liquid  above  remains  clouded 
for  some  time.  Peculiar  acid  odor. 

Streak  Cultures.— On  ayar  it  forms  a  moist,  glistening  orange-yellow 
covering.  On  potatoes  the  growth  is  excellent  forming  a  thick,  moist  yellow 
mass.  The  peculiar  odor  is  also  present. 

Bouillon.— A  slight  cl'HUi  permeates  the  liquid  and  eventually  a  yellow 
sediment  forms. 

Milk.— Coagulation  results  and  the  casein  is  then  slowly  peptonized. 

Oxygen  requirements.— Is  a  facultative  anaerobe. 
Pigment  formation  depends  on  presence  of  oxygen. 

Temperature.— Grows  at  ordinary  temperature;  best 
at  39-37°  0. 

Behavior  to  Gelatin.— Liquefies  rapidly. 

Attenuation. — The  virulence  is  rapidly  decreased  on 
artificial  media. 

Pathogenesis. — Pure  cultures  applied  to  man  pro- 
duced suppuration  and  carbuncles.  Subcutaneous  appli- 
cation in  mice,  rabbits,  and  guinea-pigs  induces  local  ab- 
scesses. Intraperitoneal  and  intravenous  injections  pro- 
duce fatal  results  with  formation  of  minute  abscesses  in 
the  different  organs  and  tissues — pyaemia.  Intravenous  in- 
joction  of  potato  cultures  induces  ulcerative  endocarditis. 
Osteomyelitis  results  when  the  bones  of  the  leg  are  first 
fractured. 

Infection. — Usually  through  scratches  and  wounds. 
May  penetrate  the  uninjured  skin. 

NOTE —In  suppuration  other  staphylococci  may  be  found,  as  the  S. 
pyogenes  albus  and  the  8.  pyogenescitreus.  These  perhaps  are  less  frequent 
and  less  virulent.  The  cultural  properties  are  nearly  the  same  with  the  ex- 
ception of  the  difference  in  pigment  production. 


MEMORANDA. 


—  168  — 
BACILLUS  PYOCYANEUS.     Gessard  (1882). 

BACILLUS    OF    GREEN    OK    BLUE    PUS. 

Origin. — In  green  pus.  The  color  forms  on  exposure 
to  air.  Several  varieties  have  been  described. 

Form. — Small  narrow  rod  resembling  that  of  blue 
milk.  At  times  it  has  almost  a  coccus  form.  The  ends 
are  rounded  ;  mny  form  short  threads  of  4-6  cells. 

Motility. — Actively  motile. 

Sporulation. — Has  not,  been  observ-ed. 

AnilinDyes. — Stains  easily  also  by  Gram's  method. 

Growth. — Is  rapid  and  abundant.  Oxygen  is  neces- 
sary to  the  formation  of  the  pigment. 

Plates.— On  gelatin  plates  a  green  fluorescing  pigment  develops  quite 
early.  The  surface  colonies  at  first  tend  to  spread  then  produce  funnel-shaped 
liquefactions.  The  deep  colonies  appear  as  round^granular  masses  with  ser- 
rated borders. 

Mich  Cultures.— In  gelatin  tubes  funnel-shaped  liquefaction  results.  The 
upper  layer  is  at  first  green  but  later  the  entire  contents  are  colored.  In  very 
old  cultures  the  color  changes  to  a  brownish  black.  A  scum  forms  on  the  sur- 
face. 

Streak  Cultures. — On  agar  a  moist,  slimy,  yellowish  growth  develops 
and  the  medium  itself  becomes  bright  green.  When  very  old  the  agar  be- 
comes dark  colored  and  the  growth  has  a  peculiar  scaly,  metallic  appearance. 
On  potatoes  a  yellowish  green  or  brownish  slimy  growth  forms. 

Milk. — Grayish  yellow  spots  form  on  the  surface;  the  casein  is  precipi- 
tated and  subsequently  peptonized  with  production  of  ammonia. 

Oxygen  requirements. — Is  a  facultative  anaerobe. 
No  growth  under  mica  plates,  but  can  grow  in  the  body. 

Temperature.— Grows  at  ordinary  temperature,  also 
in  the  incubator. 

Behavior  to  Gelatin.— Liquefies  rapidly. 

Attenuation. — Artificial  cultures  diminish  in  viru 
lence. 

Immunity.— After  recovery  from  the  effects  of  in- 
jection of  small  amounts  of  the  culture  the  animal  is 
immune.  Sterilized  cultures  also  induce  immunity. 

Pathogenesis. — Subcutaneous  injection,  in  guinea- 
pigs  and  rabbits,  of  about  1  c.c.  of  a  fresh  bouillon  culture 
produces  a  rapidly  spreading  oedema,  purulent  inflnmma- 
tion  and  death.  Bacilii  present  in  the  tissues,  blood, 
organs,  etc.  Intra  peritoneal  injections  produce  purulent 
peritonitis  and  death.  Sin*  11  amounts  produce  less  marked 
results  and  recovery.  Cure  of  animals  infected  with  an- 
thrax by  inoculation  with  B.  pyocyaneus. 


MEMORANDA. 


—  170  — 


MICROCOCCTJS  GONORRHOEA.     Neisser  (1879). 

GONOCOCCUS,  DIPLOCOCCUS  OF  GONORRIICEA. 

Origin. — Constant  in  gonorrhoea!  discharges. 

Form. — " Biscuit-shaped"  micrococci  which  are  usu- 
ally in  pairs,  with  the  flattened  surfaces  facing  each  other. 
The  diplococcus  is  usually  grouped  in  masses  of  20-40 
or  more  cells.  The  pus  cells  are  frequently  invaded  by 
the  gonococcus  which  sometimes  fills  the  cells. 

Motility.  —Has  no  real  motion. 
Sporulation. — Not  known. 

Anilin  Dyes. — Stain  readily  ;  Gram's  method  is  not 
applicable.  Methylene  blue  is  best  adapted  for  staining 
cover-glasses  of  gonorrhoea!  pus. 

Growth. — Behaves  as  a  strict  parasite.  Growth  has 
been  successfully  obtained  only  on  human  blood-serum, 
on  which  it  forms  a  very  thin,  scarcely  visible  layer  which, 
however,  soon  dies  out.  Best  results  have  been  obtained 
with  a  mixture  of  1  part  of  human  blood-serum  and  2-3 
parts  of  ordinary  nutrient  agar.  With  this  mixture  plate 
cultures  can  be  made  and  an  excellent  growth  obtained  in 
24  hours. 

Oxygen  requirements.--Apparently  it  is  an  aero- 
bic organism. 

Temperature. — Does  not  grow  below  25°,  or  above 
38°  C. 

Pathogenesis. — Pure  cultures  of  the  gonococcus, 
obtained  by  the  plate  method,  produce  typical  gonorrhoea 
when  introduced  into  the  healthy  urethra. 

NOTE.— To  examine  gonorrhoaal  pus,  covei'-glasses  should  be  prepared 
from  it  while  fresh  and  before  it  has  dried  down.  These  should  be  simple 
stained  with  methylene  blue. 


MEMORANDA, 


—  172  — 

MICROCOCCUS   TETRAGENUS. 
Koch,  Gaffky  (1881). 

Origin. — First  obtained  from  the  contents  of  a  tuber- 
cular lung  cavity;  present  in  normal  saliva  (26-times  out 
of  111  cases,  MILLER),  rather  common  in  sputum  of  tuber- 
cular persons.  Has  been  found  in  a  few  instances,  as  the 
only  organism  present  in  acute  abscesses. 

Form. — Large  cocci,  which  in  pure  cultures  on  artifi- 
cial media  are  either  single  or  in  pairs,  or  irregular  groups. 
In  the  animal  body  it  forms  perfect  tetrads,  which  are 
surrounded  by  a  wide  colorless  capsule. 

Motility.— None. 
Sporulation.— None. 

Anilin  Dyes. — Stain  readily.  Gram's  method  is 
applicable. 

Growth. — Is  rather  slow. 

Plates.— The  colonies  which  develop  on  the  gelatin  plate  are  round  or 
oval,  slightly  granular,  yellowish  and  sharp  bordered.  No  liquefaction.  The 
surface  colonies  are  white,  elevated  and  thick, 

Stich  Cultures. — Along  the  line  of  inoculation,  in  the  gelatin  tube,  the 
growth  develops  either  as  a  row  of  white  dots  or  as  a  continuous  white  line. 
On  the  surface  a  characteristic  moist,  white,  thick  mass  forms. 

Streak  Cultures.— On  agar  usually  develops  as  sharply  defined,  round, 
white  colonies.  On  potatoes  it  forms  a  thick,  slimy  covering,  which  can  be 
drawn  out  into  long  threads. 

Oxygen  requirements.— Is  aerobic,  and  also  fac- 
ultative anaerobic. 

Temperature. — Grows  well  at  ordinary  temperature ; 
better  in  the  incubator. 

Behavior  to  Gelatin. — Does  not  liquefy. 

Attenuation. — Cultures  grown  for  years  on  artificial 
media  eventually  become  attenuated. 

Pathogenesis. —  White  mice  and  guinea-pigs  are 
susceptible.  House  and  field  mice  are  usually  insuscepti- 
ble, while  rabbits  are  immune.  By  subcutaneous  applica- 
tion or  intraperitoneal  injection,  white  mice  and  guinea- 
pigs  die  in  from  3-10  days.  The  blood-vessels  of  the  kid- 
ney, spleen,  liver,  etc.,  are  full  of  the  tetrads  which  are 
invested  by  capsules. 


MEMORANDA. 


—  174  — 


SPIRILLUM    OBERMEIERI.      Obermeier  (1873). 

SPIRILLUM  OP  RELAPSING  OR  RECURRENT  FEVER  | 
SPIROCHAETE    OBERMEIERI. 

Origin. — Always  and  exclusively  present  in  the  blood 
of  relapsing  fever  patients,  especially  during  the  febrile 
paroxysms,  when  they  may  be  present  in  large  numbers. 

Form. — Delicate  flexible,  long,  wavy  spirals. 

Motility. — Actively  motile.  Flagella  have  been 
demonstrated.  Preserves  its  motility  at  ordinary  tempera- 
ture for  many  days. 

Sporulation. — Unknown. 

Anilin  Dyes. — Rapidly  and  intensely  stained  by 
ordinary  dyes. 

Growth.— Is  an  obligative  parasite.  Has  not  been 
successfully  cultivated  outside  of  the  living  body. 

Pathogenesis. — Inoculation  of  healthy  individuals 
with  blood  which  contains  these  spirals  produces  typical 
relapsing  fever,  which  is  accompanied  by  the  presence  of 
the  characteristic  spirals.  Can  also  be  transmitted  to 
monkeys,  but  the  animal  usually  recovers.  In  monkeys 
from  which  the  spleen  has  been  removed  the  spirilla 
develop  in  enormous  numbers  in  the  blood,  and  death 
results.  Although  it  has  not  been  isolated  and  grown  in 
pure  culture,  and  this,  in  turn,  tested  on  animals  yet  the 
constant  presence  of  the  organism  in  this  disease  leads  to 
the  accepted  belief  that  it  is  the  cause.  This  is  also  true 
of  the  leprosy  bacillus,  theplasmodium  of  malaria  and  the 
amaeba  coli  of  dysentery. 


MEMORANDA. 


—  176  — 

BACILLUS  OF  CHICKEN  CHOLERA. 

Perroncito,  Pasteur  (1880). 

SYNONYMS  OF  CHICKEN  OK  FOWL  CHOLERA. — CHOLERA  DES  POULES  (Fr.)  ; 
HUHNER-,  GEFLUGEL-CI10LERA   {Germ.}. 

Origin. — In  the  blood,  organs  and  excreta  of  chicken  which 
have  thejdisease.  A  somewhat  similar  disease  of  chickens,  occur- 
ing  in  Russia,  has  been  shown  to  be  due  to  the  Vibrio  Metchnikovi 
which  resembles  very  closely  the  vibrio  of  Asiatic  cholera. 

Form. — Small  short  rods  which  have  rounded  ends  and  are 
frequently  in  pairs,  rarely  in  long  threads.  At  times  the  form  is 
almost  that  of  a  coccus. 

Motility. — Has  no  motion. 

Sporulation. — Spores  have  not  been  observed.  Nevertheless, 
it  possesses  considerable  power  of  resistance  and  can  withstand  the 
acidity  of  the  gastric  juice. 

Anilin  Dyes. — Usually  stain  the  ends  first  while  the  center 
remains  uncolored — bi-polar  stain.  The  appearance  then  is  that  of 
a  diplococcus.  On  more  intense  staining  the  entire  rod  becomes 
colored.  Gram's  method  is  not  applicable. 

Growth. — Is  rather  slow. 

Plates  Colonies  appear  in  ;»  few  days  on  gelatin  plates  as  minute  white 
dots,  which,  under  the  microscope,  are  seen  to  be  roundish  plates  with  sharp, 
smooth  borders.  The  contents  ure  finely  granular,  show  concentric  rings  and 
are  yellowish  in  color.  No  liquefaction. 

Ktich  Cultures.  Forms  in  gelatin,  a  delicate  white  line  or  row  of  dots 
along  the  line  ot  inoculation.  On  the  surface  it  forms  a  delicate  whitish 
growth  which  sprctids  veiy  slowly. 

Streak  Culture*  on  aaar  it  develops  as  a  thick,  glistening,  grayish- 
white  mass.  No  growth  <>n  potatoes  at  ordinary  temperatures,  but  in  the  in- 
cubator in  u  few  tla\  s  it  gives  rise  to  a  yi  llowi^h-gray  transparent  covering. 

Oxygen  requirements.— Is  a  facultative  anaerobe. 

Temperature. — Grows  at  ordinary  temperature  and  also  in  the 
incubator. 

Behavior  to  Gelatin.— Does  not  liquefy. 

Attenuation. — Artificial  cultures  soon  lose  their  virulence.  It 
was  in  connection  with  this  organism  that  attenuation  was  first  ob- 
served by  Pasteur  (1880).  Influence  of  oxygen,  of  heat. 

Immunity. — Is  produced  in  chickens  and  pigeons  by  inocula- 
tion with  first  and  second  vaccines. 

Pathogenesis. — Chickens,  geese,  pigeons,  sparrows,  mice  and 
rabbits  are  susceptible.  Guinea-pigs,  sheep,  horses  are  less  suscep- 
tible and  only  local  abscesses  form.  After  death  the  bacilli  are  found 
distributed  throughout  the  body — a  true  septicaemia. 

Infection.  —  Usually  results  in  chicken  through  the  food 
and  along  the  alimentary  canal.  May  possibly  also  occur  through 
scratches  and  wounds. 


MEMORANDA. 


BACILLUS  OF   HOG  CHOLERA. 

Detmers  (1880). 
BILLING'S  SWINE  PLAGUE  BACILLUS.     BACTERIUM  OF  HOG  CHOLERA 

(SALMON  AND  SMITH). 

Origin. — In  the  blood,  organs  and  intestinal  contents 
of  swine  that  died  of  hog  cholera. 

Form. — Short,  small  rods,  resembling  those  of  chicken 
cholera.  On  some  media,  as  gelatin,  it  may  form  long 
rods.  Occurs  single  or  in  pairs. 

Motility. — Is  actively  motile.     Has  long,  wavy  flag- 
ella.     Shows  no  motion  in  serum  or  in  blood. 
Sporulation. — Not  observed. 

Anilin  Dyes. — At  first  impart  a  bi-polar  stain,  but 
on  sufficient  exposure  the  entire  rod   is   colored.     Is  not 
stained  by  Gram's  method. 
Growth. — Is  fairly  rapid. 

Plates.— In  a  couple  of  days  colonies  develop  on  gelatin  plates.  The 
deep  colonies  are  very  small,  yellowish-brown  and  spherical.  The  surface 
colonies  spread  slightly.  No  liquefaction. 

Stich  Culture*.— &\\o\v  along  the  line  of  inoculation  a  white  line  or  row 
of  colonies,  while  on  the  surface  of  the  gelatin  a  thin,  very  slowly  spreading 
growth  forms. 

Streak  Cultures— Qn  agar  forms  a  moist  grayish-white  growth  without 
any  special  characteristics.  On  potatoes  a  straw  yellow  growth  develops,  re- 
sembling somewhat  that  of  glanders. 

Oxygen  requirements. — Is  a  facultative  anaerobe. 

Temperature. — Grows  well  at  ordinary  temperature. 
Best  at  about  37°  0. 

Behavior  to  Gelatin. — Does  not  liquefy. 

Attenuation.— Artificial  cultures  retain  their  viru- 
lence apparently  indefinitely,  same  as  the  anthrax  bacillus. 

Immunity. — Can  be  produced  experimentally  by 
inoculation  with  filtered  cultures  ;  with  repeated  small 
doses  of  blood,  previously  heated  to  54-58°  C  ,  from  infec- 
ted rabbits. 

Pathogenesis. — Hog«,  mice,  rabbits  and  guinea-pigs 
are  highly  susceptible  ;  pigeons  are  less  susceptible,  while 
chickens,  sheep  and  calves  are  immune.  -\  c.c.  of  bouillon 
culture  injected  subcutaneously  into  rabbits  kills  in  about 
four  days.  Bacilli  distributed  everywhere. 

Infection.— May  result  through  the  food,  also  by  in- 
oculation through  wounds. 


MEMORANDA. 


—  ISO  — 


BACILLUS  OF  HOG  ERYSIPELAS. 

Pasteur  (1883). 

SYNONYMS. — SCIIWEINEKOTHLAUF   (Germ.}',    ROUGET   (Fr.). 

Origin. — In  the  blood,  internal  organs,  etc.,  of  swine  infected 
with  the  disease. 

Form. — Very  small,  narrow  rods  resembling  needle-shaped 
crystals.  Are  usually  single,  but  may  occur  in  pairs  and  even  in 
threads. 

Motility. — Has  motion. 

Sporulation. — Spore  formation  is  not  known. 

Anilin  Dyes.— Stain  readily.  Gram's  method  gives  excellent 
results. 

Growth  — Is  rather  slow. 

Growth.— On  gelatin  plates  the  colonies  are  very  characteristic  and  ap- 
pear as  diffuse  cloudy  patches  which  are  sometimes  difficult  to  see.  Little 
or  no  surf  are  growth.  No  liquefaction 

Stick  Cultures.— In  gelatin  are  likewise  very  characteristic.  The  growth 
develops  along  the  line  of  inoculation  as  a  delicate,  cloud-like  radiating  col- 
umn. As  the  culture  becomes  old  a  depression  forms  at  the  top,  due  to  slow 
liquefaction  and  corresponding  evaporation.  .Sometimes  liquefaction  can 
be  observed. 

Streak  Cultures. — On  agar  and  on  blood- serum  it  forms  a  scarcely  visible 
thin  film  or  group  of  colonies.  No  growth  on  potatoes. 

Bouillon.— A  very  delicate  diffuse  cloudiness  forms  which  can  best  be 
seen  on  slight  agitation.  Resembles  the  bouillon  culture  of  the  Tetanus 
bacillus. 

Oxygen  requirements. — Is  a  facultative  aerobe.  Best  growth 
under  anaerobic  conditions. 

Temperature. — Grows  slowly  at  ordinary  temperature.  Best 
at  36°  C. 

Behavior  to  Gelatin. — Does  not  perceptibly  liquefy  gelatin. 

Aerogenesis  — Produces  hydrogen  sulphide  in  pure  cultures, 
and  in  the  body.  This  gas  is  also  produced  by  the  anaerobic  bac- 
teria and  to  a  less  extent  by  nearly  all  pathogenic  bacteria. 

Attenuation  — Old  cultures  become  attenuated  and  this  re- 
sult can  also  be  obtained  by  growing  the  virulent  germ  at  high  tem- 
peratures, about  42°  C.,for*some  time  (Pasteur). 

Immunity. — By  inoculation  with  attenuated  cultures — first 
and  second  vaccine  of  Pasteur — perfect  immunity  can  be  produced. 
One  attack  of  the  disease  confers  immunity. 

Pathogenesis. — Swine,  rabbits,  pigeons, white  mice,  house  mice 
'are  susceptible,  while  guinea-pigs  and  chickens  are  insusceptible. 
Bacilli  distributed  throughout  the  organism  ;  are  single  or  in  pairs, 
and  very  often  can  be  seen  to  be  enclosed  in  cells. 

Infection. — Probably  occurs  naturally  in  swine  through  the 
food. 


MEMORANDA. 


—  182  ~ 


BACILLUS  OF  MOUSE  SEPTIC^MIA. 
Koch  (18T8J. 

SYNONYMS. — BACILLUS  MURISEPTICUS.       MAUSESEPTIKAMIE    (Germ.). 

Origin. — From  mice  after  inoculation  with  putrid  blood. 

Form. — The  rods  are  narrower  and  thinner  than  those  of  the 
rouget  bacillus,  but  otherwise  resemble  the  latter  very  much. 

Motility. — Appears  to  possess  motion.  Said  to  be  non-motile 
by  some. 

Sporulaticn. — Round,  glistening  bodies,  or  spores  form  within 
the  cells. 

Anilin  Dyes. — Stain  rapidly.     Gram's  method  is  applicable. 

Growth. — Is  rather  slow  and  resembles  very  closely  that  of 
the  rouget  bacillus. 

Plates.— The  colonies  on  the  gelatin  plate  resemble  those  of  the  rouget 
bacillus,  except  that  they  spread  somewhat  more  rapidly  and  are  especially 
delicate  and  transparent  in  appearance. 

Stich  Cultures.— Show  this  distinction  in  growth  quite  sharply.  While 
the  cloudy  growth  of  the  rouget  bacillus  is  dense  and  somewhat  limited  to  the 
line  of  inoculation,  that  of  the  mouse  septicaemia  bacillus  spreads  readily 
throughout  the  entire  gelatin.  This  difference  is  clearly  seen  in  young 
cultures. 

Streak  Cultures. — On  agar  the  growth  is  scarcely  to  be  distinguished 
from  that  of  the  rouget  bacillus. 

Bouillon  — The  bacillus  develops  a  growth  similar  to  that  of  the  bacillus 
of  rouget. 

Oxygen  requirements. — Is  a  facultative  aerobe.  Grows  better 
when  air  is  excluded. 

Temperature. — Grows  well  at  ordinary  temperature,  also  in 
the  incubator. 

Behavior  to  Gelatin. — Ordinarily  no  liquefaction  can  be  ob- 
served. Sometimes,  however,  it  is  present. 

Aerogenesis. — Produces  less  hydrogen  sulphide  than  the  rou- 
get bacillus. 

Attenuation  — Old  cultures  possess  diminished  virulence. 

Immunity. — Rabbits  that  recover  after  one  inoculation  with 
the  pure  culture  are  rendered  immune  against  subsequent  inocula- 
tion. 

Fathogenesis. — White  mice,  house  mice,  pigeons,  sparrows 
and  rabbits  are  susceptible.  Chickens,  guinea-pigs  and  field  mice  are 
wholly  immune.  After  death  the  bacilli  are  distributed  throughout 
the  body,  single  or  in  pairs,  and  frequently  inclosed  in  cells. 


MEMORANDA. 


16 


—  184 


ACTINOMYCES.    Bellinger  (1877). 

SYNONYMS. — RAY-FUNGUS.       STRAIILENPILZ   (Germ.}. 

Origin. — From  actinomycosis  or  lumpy-jaw  in  cattle, 
hogs,  and  in  man. 

Form. — The  exact  position  of  this  organism  is  uncer- 
tain, but  it  is  closely  related  to  the  fungi  or  moulds.  It 
forms  nodules  which  consist  of  a  whorl  of  mycelial-like 
multiple  branched  threads.  These  radiate  outward  from 
a  central  point  and  become  club-shaped.  In  pure  cultures 
only  slender,  wavy  threads  are  formed,  and  the  club-shaped 
or  swollen  ends,  so  commonly  present  in  tissues  are  lacking. 

Anilin  Dyes.— Stain  readily  with  carbolic  fuchsine; 
also  by  Gram's  method. 

Growth. — Develops  somewhat  slowly,  requiring  sev- 
eral days  in  the  incubator. 

Streak  Cultures. — On  agar,  the  growth  begins  as  minute,  isolated  colo- 
nies, which  slowly  enlarge,  forming  thick,  convex,  glistening,  yellowish 
masses.  These  colonies  are  exceedingly  hard  and  for  examination  should  be 
crushed  between  two  glass  slides  previously  sterilized  by  passing  several  times 
through  the  flame.  Cover-glass  preparations  are  then  made  and  stained  in 
the  usual  manner. 

Oxygen  requirements.— Said  to  grow  best  in  the 
absence  of  air,  but  grows  very  well  on  the  surface  of  agar. 

Temperature. — Grows  only  at  or  near  the  body 
temperature. 

Pathogenesis. — In  rabbits,  intraperitoneal  injection 
of  the  pure  culture  produces  typical  actinomycotic  nodules 
on  the  peritoneum,  mesentery,  intestinal  walls,  etc. 


MEMORANDA. 


—  186  — 


ACHORION  SCHONLEINII.     Schonlein  (1839). 

THE  FUNGUS    OP   FAVUS. 

Origin. — Found  in  the  scaly  accumulations  on  the 
skin  of  persons  afflicted  with  favus. 

Form. — Apparently  belongs  to  the  moulds.  It  shows 
on  microscopical  examination  peculiarly  twisted  threads, 
which  show  divisions  and  give  off  branches  at  right  angles. 

Fruit-organs.— No  true  fruit  organs  observed,  but 
on  special  media  as  on  blood-serum  at  30°  0.  conidia  or 
spores  form. 

Anilin  Dyes.— Stains  well,  also  by  Gram's  method. 

Growth. — Is  rather  slow. 

Plates.— On  gelatin  plates,  the  colonies  grow  slowly  and  form  whitish, 
stellate  masses,  which  rapidly  liquefy  the  gelatin.  No  conidia  present. 

Slich  Cultures.— Growth  is  very  poor  in  the  lower  part  of  the  gelatin 
tube.  On  the  surface  it  forms  a  white  covering,  the  lower  side  of  which  is 
light  yellow.  Liquefies. 

Streak  Cultures.— On  agar  it  forms  a  closely  adherent,  whitish,  dry  mass. 

Temperature. — Dies  out  at  the  ordinary  tempera- 
ture. The  optimum  is  about  30°  C. 

Behavior  to  Gelatin.— Liquefies. 

Pathogenesis.— Inoculation  with  pure  culture  pro- 
duces typical  favus  in  man. 

The  favus  fungus  is  closely  related  to  that  of  Herpes 
tonsurans — the  Tricophyton  tonsurans  (1845);  to  that  of 
Pityriasis  versicolor— the  Microsporon  furfur  (1846);  and 
also  to  the  Oidium  lactis. 


MEMORANDA. 


—  188  — 


MONILIA  CANDIDA.     Robin  (1847). 

SYNONYMS. — THRUSH    FUNGUS;    OIDIUM  ALBICANS,  SACCHAROMYCES 

ALBICAXS.     SOORPILZ  (Germ.}. 

Origin. — Found  in  the  mouths  of  infants  ;  in  thrush. 

Form. — Occupies  an  intermediate  position  between 
the  moulds  and  yeasts.  On  gelatin  plates  and  on  sugar 
media  it  forms  yeast-like  cells,  whereas  in  the  deeper 
part  of  the  stich  culture  it  forms  mycelial  threads. 

Anilin  Dyes.— Stain  readily. 

Growth. — Is  rapid  and  abundant. 

Plates.— Snow-white  colonies  form  on  gelatin  plates  and  no  liquefaction 
takes  place. 

Stich  Cultures.— In  gelatin  show  growth  along  the  line  of  inoculation, 
while  on  the  surface  a  milk-white,  thick  mass  forms. 

Streak  Cultures. — On  agar,  forms  a  glistening,  moist,  thick,  white 
growth.  On  potatoes,  it  grows  rapidly  as  a  thick,  white,  yeast-like  mass. 

Temperature. — Grows  at  ordinary  temperature,  also 
in  incubator. 

Behavior  to  Gelatin. — Does  not  liquefy. 

Pathogenesis.  —  Intravenous  injection  in  rabbits 
produces  death  in  1-2  days.  The  internal  organs  are  per- 
meated with  a  growth  of  long  mycelial  threads. 


MEMORANDA. 


MEMORANDA. 


MEMORANDA. 


MEMORANDA. 


—  189  — 


SPECIAL  WORK. 


Direct  microscopical  examination  of  streak  prepara- 
tions made  from  the  organs  and  tissues  of  infected  ani- 
mals, as  well  as  cultural  experiments  will  reveal  the  pres- 
ence of  microorganisms.  In  order  to  ascertain  the  presence 
and  especially  the  distribution  of  organisms  within  tissues 
and  organs  it  is  necessary  to  harden  these,  then  to  cut 
sections  and  finally  to  stain  the  sections  by  suitable  meth- 
ods. 

Hardening.— For  this  purpose  alcohol  is  usually 
employed  and  gives  excellent,  results.  The  tissue  is  cut 
into  small  pieces  which  are  either  transferred  direct  to  a 
wide-mouthed  bottle  containing  92-96  per  cent,  alcohol,  or 
are  first  placed  on  pieces  of  filter  paper  and  then  in  the 
alcohol.  The  pieces  of  tissue  should  remain  in  this  alcohol 
for  at  least  3  or  4  days,  or  until  it  is  desired  to  make  sec- 
tion?, when  they  are  transferred  to  absolute  alcohol  for 
one  or  two  days.  The  tissue  is  then  hardened  and  ready 
for  cutting  sections.  To  do  tljis  a  piece  of  the  tissue  is  at- 
tached to  a  small  cork  by  means  ot  a  glycerine  gelatin 
mixture  made  by  warming  1  part  of  gelatin,  2  parts  of 
water  and  4  parts  of  glycerine.  The  cork  is  then  securely 
clamped  to  the  microtome  and  sections  made.  The  tissue 
and  knife  must  be  kept  moist  with  alcohol  and  the  sec- 
tions are  at  once  transferred  to  alcohol  by  means  of  a 
camel  hair  brush. 

Very  satisfactory  and  rapid  hardening  can  be  obtained 
with  a  solution  of  mercuric  chloride  made  by  saturating 
an  aqueous  five  per  cent,  glacial  acetic  acid  solution  with 
mercuric  chloride.  The  tissue  can  be  fixed  in  this  solution 
in  4  to  12  hours.  It  is  then  passed  through  a  series  of 


—  190  — 

alcohols   of  different  strength, — 60,  80,  96X  and  absolute, 
in  each  of  which  the  tissue  remains  for  24  hours. 

Cutting  sections. — As  already  stated  the  tissue 
which  has  been  hardened  in  alcohol  may  be  cut  directly 
and  sections  can  thus  be  obtained  which  are  fairly  good. 
Another  method  for  obtaining  sections  is  to  employ  the 
freezing  microtome,  in  which  case  the  alcohol  is  first  re- 
moved from  the  tissue  by  placing  the  pieces  in  water. 
This  is  accomplished  in  cold  water  in  4-8  hours,  depend- 
ing: on  the  size  of  the  pieces.  Warm  water  about  38°  C. 
will  remove  the  alcohol  more  rapidly,  in  1  to  2  hours. 
The  tissues  can  then  be  frozen  and  sections  cut.  The 
knife  should  be  kept  moistened  with  water  and  the  sec- 
tions are  transferred  at  once  to  water. 

Undoubtedly  the  most  satisfactory  method  of  prepar- 
ing sections  is  to  first  imbed  the  hardened  tissue  either  in 
celloidin,  or  in  paraffin.  Either  method  gives  excellent 
results.  The  method  for  imbedding  in  paraffin  which  is 
usually  employed  in  the  laboratory  is  briefly  as  follows : 

The  hardened  tissue  is  placed  in  absolute  alcohol  for 
24  hours;  then  for  4  to  5  hours  in  chloroform,  then  in  a 
chloroform-paraffin  solution  over  night.  The  pieces  are 
then  placed  in  paraffin  which  melts  at  about  42°  C.  and  kept 
in  a  water  air-bath  at  a  temperature  of  about  50°  for  24 
hours.  From  this  the  tissue  is  transferred  to  hard  paraffin 
which  melts  at  about  48°  C.  This  may  be  obtained  by 
taking  equal  parts  of  42°  and  56°  paraffin.  After  24 
hours  the  piece  of  tissue  is  "blocked.-'  .  Two  glass  L's  are 
fitted  together  so  as  to  make  a  trough  of  suitable  size,  and 
this  then  filled  with  melted  paraffin  (48°  C.)  The  piece  of 
tissue  is  transferred  by  means  of  a  pair  of  forceps,  slightly 
warmed,  to  the  center  of  the  block  and  the  whole  allowed 
to  cool.  The  solid  block,  if  necessary,  is  then  trimmed, 
fastened  to  the  microtome  and  sections  cut  with  a  dry 
knife. 

From  absolute  alcohol  the  pieces  of  tissue  can  be 
placed  first  in  toluene  for  24  hour,s;  then  in  a  mixture  of 


MEMORANDA. 


MEMORANDA. 


—  191  — 

equal  parts  of  toluene  and  paraffin  for  24  hours  and  then 
in  paraffin  for  24-  hours. 

The  paraffin  can  be  dissolved  from  the  sections  by 
means  of  xylol  or  turpentine.  The  sections  are  then  placed 
in  absolute  alcohol  and  finally  transferred  to  10%  alcohol, 
in  which  they  may  be  kept  for  any  length  of  time  and  are 
now  ready  for  staining, 

Paraffin  sections  sometimes  tend  to  curl  or  become 
folded.  This  difficulty  can  be  readiiy  overcome  by  plac- 
ing the  sections  in  a  Petri  dish  containing  tepid  water. 
This  must  not  be  so  warm  as  to  melt  the  paraffin.  The 
dish  may  be  kept  on  an  iron  plate  which  is  heated  gently 
at  one  end.  The  sections  spread  out  on  the  surface  of  the 
warm  water.  They  can  be  received  on  strips  of  paper  and 
transferred  to  a  glass-slide  or  cover-glass  which  is  covered 
with  a  film  of  albumin.  The  section  is  then  dried  with  a 
piece  of  paper  and  caused  to  adhere  by  slightly  warming 
the  glass  slide.  It  can  then  be  stained  in  the  usual  man- 
ner. The  method  is  very  convenient  in  working  with  very 
thin  sections  or  with  very  delicate  tissues.  (BORREL). 

Staining  of  Sections. — The  presence  of  organisms 
in  sections  of  tissues  is  sometimes  very  difficult  to  demon- 
strate although  they  may  be  easily  shown  to  be  present 
in  ordinary  streak  cover-glass  preparations.  This  is  fre- 
quently due  to  the  absence  of  any  sharp  means  of  differ- 
entiating the  organism  from  the  surrounding  tissue.  In 
many  cases,  however,  a  sharp  differentiation  can  be  ob- 
tained by  double  staining  either  by  Gram's  method,  or,  as 
in  the  case  of  leprosy  and  tuberculosis,  by  the  Triplication 
of  the  usual  process  for  staining  these  bacilli. 

Simple  stain. — The  student  should  begin  with  cec- 
lions  of  the  spleen,  kidney  and  liver  of  a  guinea-pig  which 
died  of  anthrax.  The  sections  are  transferred  by  means 
of  a  needle  to  the  dilute  anilin  .dye,  as  fuchsine  or  gentian 
violet  and  are  allowed  to  remain  there  for  10  to  30 
minutes.  They  are  then  transferred  to  water  slightly  acid- 
ulated with  acetic  acid,  or  to  very  dilute  alcohol.  This  is 


—  192  — 

done  to  remove  the  excess  of  dye  and  to  differentiate  the 
bacteria  in  the  tissue.  From  the  decolorizing  solution  the 
section  is  transferred  to  water  then,  by  means  of  a  spa- 
tula, to  a  glass  slide  and  examined  with  a  No.  7  objective. 
If  the  section  is  still  too  intensely  stained  it  should  be  re- 
turned to  the  decolorizing  agent  and  after  a  while  again 
examined.  When  satisfactorily  stained  the  sections  should 
be  placed  in  absolute  alcohol,  for  a  few  seconds,  till 
thoroughly  dehydrated,  they  are  then  cleared  up  in  oil  of 
cloves,  cedar  or  anise;  placed  in  xylol  and  examined  on 
a  slide.  If  satisfactory  the  cover-glass  is  carefully  lifted 
off  and  the  xylol  removed  from  the  section  by  means  of  a 
piece  of  filter  paper.  A  drop  of  Canada  balsam  is  then 
applied  to  the  section  and  the  whole  covered  with  a  clean 
cover-glass.  The  exposure  to  absolute  alcohol  and  to  oil 
of  cloves  should  be  carefully  watched  as  both  tend  to  re- 
move the  stain.  The  oil  of  cloves  can  indeed  be  relied 
upon  to  remove  any  excess  of  dye  that  may  be  present. 

Instead  of  the  ordinary  dilute  anilin  stain,  Lo  (Tier's 
methylene  blue  (page  140)  or  ZiehFs  carbolic  fuchsine 
(page  110)  can  be  employed  in  special  cases  to  excellent 
advantage.  A  dilute  ZiehTs  solution  gives  particularly 
good  results.  The  sections  remain  in  this  for  about  half  an. 
hour  and  are  then  transferred  to  absolute  alcohol  which  is 
very  slightly  acidulated  with  acetic  acid.  As  soon  as  the 
color  changes  to  a  peculiar  reddish  violet  tint  the  section 
is  removed,  cleared  up  in  xylol,  examined  and  mounted  in 
balsam.  (Pfeiffer's  method.) 

Double  stain — Gramas  method. — Those  microorgan- 
isms which  can  be  stained  by  this  method  can  be  readily 
detected  in  sections,  as  the  preparations  when  properly 
made  show  the  heavily  stained  violet  bacilli  on  a  light  pink 
background.  The  student  should  begin  with  sections  of 
the  kidney  of  the  anthrax  guinea-pig.  A  strong  solution 
of  anilin  water  gentian  violet  is  prepared  according  to  the 
directions  given  on  page  106.  It  should  be  warmed  slightly 
on  the  radiator,  or  on  an  iron  plate.  The  sections  are  placed 


MEMORANDA. 


MEMORANDA. 


—  193  — 

in  this  slain  for  15  to  30  minutes.  They  are  then  washed 
in  anilin  water  to  remove  excess  of  the  dye,  and  thus  to 
prevent  the  formation  of  unsightly  deposits  on  subsequent 
contact  with  iodine.  The  sections  are  then  placed  in  the 
solution  of  iodine  in  potassium  iodide  (p.  107)  for  2  or  3 
minutes.  From  this  they  are  transferred  to  absolute  alco- 
hol and  gently  moved  about  till  most  of  the  stain  is  re- 
moved. The  sections  should  be  still  slightly  stained,  not 
completely  decolored.  They  are  then  placed  in  Weigert's 
picrocarmine  solution  or  in  eosine,  for  1-2-3  minutes,  de- 
hydrated in  absolute  alcohol  for  a  few  seconds  and  then 
transferred  to  oil  of  cloves  in  which  the  sections  are  al- 
lowed to  remain  till  all  the  gentian  violet  lias  been  re- 
moved. Oil  i/f  cloves,  especially  when  dark  colored,  has 
strong  decolorizing  properties  and  will  remove  all  traces 
of  gentian  violet  from  the  tissues  without  affecting  the 
bacilli  to  any  extent.  The  sections  are  then  placed  jn 
xylol  and  examined  and  if  satisfactory  mounted  in  Can- 
ada balsam. 

ANTHRAX    SECTIONS. 


Simple  Stain:  Gra'tns   Method: 

Dilute  anilin  stain  Anilin  water-gentian  violet 

(10  to  30  min.).  (warirr  15  to  30  min.). 

Acetic  water.  Iodine  in  potassium  iodide 

Water  (and  exam-  (2-3  min.). 

ine).  Absolute  alcohol. 

Absolute  alcohol  Picrocarmine  (1-2-3  min.). 

(few  seconds).  Absolute  alcohol    (few 

Oil  of  cloves.  seconds). 

Xylol  (and  exam-  Oil  of  cloves  (till  violet 

ine).  ceases  to  be  given  off). 

Canada  Balsam.  Xylol  (and  examine). 

Canada  balsam. 

Malignant  oedema.— Prepare  sections  from  the  ab- 
dominal wall,  kidney  and  liver  of  a  guinea-pig  which  died 
after  inoculation  with  the  bacillus  of  malignant  oedema. 


—  194  — 

The  tissues  and  organs  should  be  removed  48  hours  after 
the  death.  Stain  with  dilute  carbolic  fuchsine  according 
to  Pfeiffer's  method  as  given  on  page  192. 

Symptomatic  anthrax.— Prepare  sections  from  the 
same  tissues  as  above  from  a  guinea-pig  which  was  not  ex- 
amined till  48  hours  after  the  death.  Stain  by  the  same 
method  and  compare  the  two  organisms. 

Bacillus  cedematis  maligni,  No.  II.— Section  the 
thickened  abdominal  wall  and  stain  after  Gram's  method. 

Tubercle  bacillus. — Prepare  sections  of  tubercular 
human  lung,  also  of  the  spleen,  liver,  and  mesenteric  tu- 
bercles of  a  guinea-pig  inoculated  with  tubercular  spu- 
tum. Stain  the  sections  according  to  the  following  method 
which  is  a  modification  of  the  Ziehl-Neelsen  method. 

The  sections  are  placed  in  Ziehl's  carbolic  fuchsine, 
slightly  warmed,  for  15-30  minutes.  They  are  then  trans- 
ferred to  Ebner's  solution  where  they  are  moved  about 
till  the  color  ceases  to  be  given  off.  The  sections  should  still 
possess  a  slight  pink  color.  They  are  then  placed  in  dilute 
methylene  blue  for  ^-1  minute.  From  this  they  are  trans- 
ferred by  means  of  a  spatula  to  absolute  alcohol  for  -J-l 
minute.  The  sections  must  not  remain  in  the  alcohol  till 
all  the  blue  disappears.  They  are  then  placed  in  oil  of 
anise,  transferred  to  xylol  and  examined.  If  satisfactory 
the  section  is  mounfed  in  Canada  balsam. 

Ebner's  decalcifying  solution  is  prepared  according  to 
the  formula: — Sodium  chloride  0.5,  hydrochloric  acid  0.5, 
alcohol  100,  distilled  water  30. 

Instead  of  using  Ebner's  solution  for  decoloring  the  tis- 
sues a  2  per  cent,  aqueous  solution  of  anilin  hydrochloride 
can  be  employed  with  excellent  results  as  it  has  little  or 
no  tendency  to  decolor  the  tubercle  bacilli.  (KiiHNE, 
BORREL).  The  sections  are  stained  in  Ziehl's  solution  as 
above,  then  placed  for  a  few  seconds  in  the  2  per  cent, 
aqueous  solution  of  anilin  hydrochloride,  then  washed  in 
alcohol  and  counter-stained  as  above. 

Leprosy  bacillus.— Sections  of  the  skin  of  a  leper 


MEMORANDA. 


MEMORANDA. 


—  195  — 

can  be  double  stained  according  to  the  method  described 
for  the  tubercle  bacillus.  It  can  be  summarized  as  fol- 
lows : 

Sections. 

Carbolic  fuchsine  (warm  15-30  min.). 

Ebner's  solution  (till  it  turns  a  light  pink). 

Dilute  methylene  blue  (-J— 1  min.), 

Absolute  alcohol  (-J-1  min.). 

Oil  of  anise. 

Xylol  (and  examine). 

Canada  balsam. 

Leprosy  sections  left  in  dilute  alcohol  soon  lose  their 
capacity  for  double  staining.  They  can  be  simple  stained 
with  methylene  blue,  or  with  carbolic  fuchsine  by  Pfeif- 
fer's  method. 

Glanders  bacillus.— The  detection  of  this  bacillus 
in  tissue  i?  rather  difficult  owing  to  its  marked  peculiarity 
of  readily  becoming  decolorized.  Sactions  from  the 
spleen,  of  a  guinea  pig  should  be  simple  stained  with 
Lo filer's  alkaline  meihylene  blue  (p.  140)  or  with  carbolic 
fuchsine. 

Typhoid  fever  bacillus. —The  E berth  bacillus  al- 
though it  is  siained  readily  and  intensely  is  very  likely 
to  become  decolorized  in  the  ordinary  method  of  staining. 
Sections  of  human  spleen  are  stained  in  Loffler's  alkaline 
methylene  blue  for  24  hours.  Then  washed  and  decolored 
in  water,  dehydrated  in  anilin  oil,  allowed  to  dry  on  a  slide 
and  finally  cleared  up  with  xylol.  Simple  stains  can  be 
made  with  carbolic  fuchsine,  decolorizing  carefully  in  acid- 
water  and  alcohol. 

Frankel's  diplococcus.— Sections  from  the  lung, 
spleen,  liver  etc.,  of  a  rabbit  can  be  stained  by  Gram's 
method, 

Loffler's  diphtheria  bacillus. — Sections  of  diph 
thentic  membranes,   or  of  muscles  from  the  neighborhood 
of  the  point  of  inoculation  should  be  stained  with  Loffler's 
alkaline  methylene  blue,  or  by  Gram's  method. 


—  196  — 

Staphylococcus  pyogenes  aureus.-  This  organism 

also  the  Streptococcus  pyogenes  can  be  detected  in 
sections  of  the  kidney,  suprarenal  body  etc.  in  pyaemia 
of  man,  or  in  the  organs  of  rabbits  inoculated  with  pure 
Cultures.  The  sections  should  be  stained  by  Gram's 
method,  or  with  carbolic  fuchsine. 

Chicken  cholera  bacillus.— This  can  best  be  de- 
monstrated in  sections  of  the  liver,  spleen  and  pectoral 
muscles  of  a  pigeon.  Simple  staining  with  carbolic  fuch- 
sine or  anilin  water  gentian  violet  will  give  fair  results. 

Micrococcus  tetragenus. — The  kidneys,  lungs  etc. 
•of  white  mice  and  guinea-pigs  give  excellent  preparations 
when  stained  by  Gram's  method. 


MEMORANDA. 


OF  THE 

(  UNIVERSITY   ) 

OF 


MEMORANDA. 


—  197 


TESTING  OF  DISINFECTANTS. 

In  studying  the  action  of  physical  and  chemical  agents 
on  bacteria  it  is  necessary  to  rigidly  adhere  to  certain  re- 
quirements without  which  the  results  would  be  of  little 
value,  if  not  wholly  contradictory.  The  conditions  which 
underly.the  testing  of  disinfectants  may  be  summed  up  as 
follows. 

(1 ).  Variable  resistance  of  spores  and  of  the  vegetat- 
ing forms  of  one  and  the  same  organism.  It  has  been 
shown  in  recent  years  that  considerable  variation  may  exist 
in  the  resistance  which  an  organism  possesses  to  destruc- 
tion. Thus,  while  there  are  some  spores  of  anthrax  which 
are  readily  destroyed  by  steam-heat,  100°  C.,  others  have 
been  known  to  withstand  this  temperature  for  10-12  min- 
utes. Again  it  was  formerly  stated  that  anthrax  spores 
were  destroyed  by  5  per  cent,  carbolic  acid  in  two  days 
but  the  researches  of  Fraenkel  have  shown  that  spores  of 
anthrax  may  be  had  which  are  not  destroyed  by  an  ex- 
posure of  30  to  40  days.  In  view  of  these  facts  several 
standards  have  been  proposed.  Thus  Fraenkel  designates 
anthrax  spores  which  are  destroyed  by  5  per  cent,  carbolic 
in  less  then  10  days  as  feebly  resistant;  in  10  to  20  days 
as  of  average  resistance;  in  20  to  30  days  as  very  resistant; 
in  30  to  40  days  as  extremely  resistant.  Geppert's  stand- 
ard an fchiMX  sp)t35rir  e  those  which  are  infectious  after 
boiling  for  one  minute  1  c.  c.  of  a  spore  suspension  which 
is  added  to  30  c.  c.  of  boiling  water.  Esmarch  has  sug- 
gested as  a  standard  anthrax  spores  which  when  fixed  on 
silk  threads  resist  steam-heat  of  100°  G.  for  10  minutes. 

(2).  The  influence  of  the  medium  in  which  the  organ- 
ism is  tested.  Thus  it  has  been  shown  that  to  destroy  an- 
thrax spores  in  bouillon  it  requires'20  tin)3s  as  much  mer- 


—  198  — 

curie  chloride  (1-1000)  than  when  they  are  suspended  in 
water,  and  250  times  as  much  when  they  are  distributed 
in  blood  serum. 

(3).  The  temperature  at  which  the  disinfection  is 
made.  The  higher  the  temperature  at  which  the  experi- 
ments are  made  the  more  rapid  and  energetic  will  be  the 
action  of  the  disinfectant.  Cholera  bacteria  are  not  de- 
stroyed bymercuric  chloride  (1-1000)  in  one  hour  at  —3°, 
whereas  at  36°  C.  they  are  killed  in  a  few  minutes. 

(4).  Immediate  and  thorough  contact  of  all  the  or- 
ganisms present  with  the  disinfectant.  This  can  be  done 
perfectly  only  with  bacterial  suspensions  in  which  each  or- 
ganism is  entirely  free  and  separate  from  others.  To  ob- 
tain such  a  suspension  it  is  necessary  first  to  filter  through 
glass  wool  and  then  to  agitate  the  liquid  thoroughly  at  a 
temperature  of  about  37°  until  microscopical  examination 
shows  no  aggregations  of  bacteria.  Silk  threads  which  have 
been  soaked  in  bacterial  suspensions  and  then  dried  are 
open  to  the  objection  that  on  exposure  to  the  disinfectant 
organisms  are  unequally  exposed  and  some  even  protected 
by  their  position  and  hence  when  transplanted  soon  de- 
velop. The  same  objection,  to  a  less  degree,  applies  to 
cover-glasses  on  which  a  thin  film  of  the  suspension  has 
been  deposited. 

(5).  The  number  of  bacteria  in  a  given  experiment. 
It  can  readily  be  shown  that  the  greater  the  number  of 
bacteria  present  the  more  slowly  does  the  disinfection 
take  place.  In  order  therefore  that  results  may  be  com- 
parable, approximately  the  same  number  of  organisms 
should  be  present  in  each  experiment.  This  is  readily  as- 
certained by  diluting  a  small  portion  of  the  bacterial  sus- 
pension witli  1-2000  parts  of  sterilized  water  and  then 
making  a  gelatin  plate  with  one  drop  of  this  dilution. 

(6).  The  amount  of  the  disinfectant  which  is  carried 
over  in  each  trial  inoculation.  Thus,  when  the  disinfect- 
ant is  applied  to  the  bacterial  suspension  and  at  the  end 
of  stated  intervals  transfers  of  1-3  loopfuls  of  the  mixture 


MEMORANDA. 


MEMORANDA. 


—  199  — 

are  made  to  sterilized  nutrient  media,  a  sufficient  amount 
of  the  disinfectant  may  be  carried  over  to  prevent  the 
growth  of  the  organism  which  mny  still  possess  vitality. 
This  has  been  a  most  serious  source  of  error  in  the  past. 
The  error  is  more  marked,  the  greater  the  antiseptic 
power  of  the  disinfectant.  It  is  of  course  less  marked 
where  the  substance  has  weak  antiseptic  properties 
and  where  the  transplantation  occurs  into  relatively 
large  amounts  of  the  nutrient  medium  (10  to  15  c.  c.). 
It  must  be  remembered  that  probably  in  all  cases  the 
first  action  of  a  disinfectant  is  to  attenuate  the  organism 
and  that  when  the  latter  is  in  this  condition  a  much 
smaller  amount  of  the  disinfectant  will  act  as  an  antiseptic 
and  prevent  growth.  This  has  been  especially  shown  to 
be  the  case  with  reference  to  the  action  of  mercuric  chlo- 
ride on  anthrax  spores.  Formerly  it  was  disposed  that 
these  were  killed  by  this  substance  in  a  strength  of  1  to 
1000  in  one  minute  but  if  the  mercury  which  is  held  fast 
by  the  silk  thread,  and  which  cannot  be  removed  by  mere 
washing,  is  removed  by  the  action  of  hydrogen  sulphide  it 
can  be  shown  that  the  organism  is  alive  and  infectious 
even  alter  an  exposure  of  four  hours.  It  may  even  possess 
vitality  alter  an  exposure  of  24  hours.  The  first  action  of 
the  disinfectant  in  this  instance  is  to  attenuate  the  organ- 
ism the  growth  of  which  is  then  prevented  by  mere  traces 
of  the  mercury.  One  part  in  two  million  according  to 
Geppert  suffices  to  produce  this  result. 

(7).  Observation  of  the  trial  inoculation  tubes  over  a 
considerable  length  of  time.  The  failure  of  tubes  to  grow 
within  24  hours  is  not  a  positive  indication  that  the  organ- 
ism has  been  destroyed  by  the  disinfectant.  In  the  at- 
tenuated condition  the  organism  will  grow  much  more 
slowly  then  it  would  if  normal  and  in  possession  of  full  vi- 
tality. Moreover,  as  stated  already,  traces  of  the  disin- 
fectant which  are  carried  over  in  the  experiment  will  still 
further  tend  to  retard  the  growth.  For  these  reasons  the 


—  200  — 

tubes  should  be  kept  under  observation  for  1 — 2 — 3  weeks 
before  definite  conclusions  can  be  drawn. 

(8).  Temperature  at  which  the  trial  inoculation 
tubes  are  kept.  The  organism  which  has  been  exposed  to 
the  action  of  the  disinfectant  should  be  placed  under  con- 
ditions which  are  most  favorable  to  its  growth.  That  is, 
the  best  nutrient  medium  and  the  most  suitable  tempera- 
ture should  be  furnished.  Transplantations  made  into  gel- 
atin and  kept  at  ordinary  room  temperature  frequently 
fail  to  grow  while  parallel  bouillon  and  agar  cultures, kept  in 
the  incubator,develop.  It  is  therefore  desirable  to  make  the 
transplantation  onto  the  surface  of  inclined  agar  tubes  or 
into  bouillon  and  to  keep  the  tubes  under  observation  at  a 
temperature  of  about  37.5°  C.  for  two  or  three  weeks. 

(9).  Negative  experiments  with  animals  inoculated 
with  organisms  exposed  to  heat,  or  to  the  action  of  chemi- 
cals prove  nothing.  The  organism  may  be  dead  or  it  may 
have  become  attenuated  and  is  therefore  without  action, 
although  it  may  still  grow  on  artificial  media.  Thus,  an- 
thrax spores  exposed  to  the  boiling  temperature  for  2  min- 
utes no  longer  kill  guinea-pigs,  but  nevertheless  can  grow 
in  tubes,  even  after  5  minutes  exposure.  Again,  positive 
experiments  may  be  obtained  by  inoculating  white  mice  or 
guinea-pigs  with  the  mixture  of  bacteria  and  disinfectant  at 
a  time  where  transplantation  of  a  corresponding  amount  on 
a  nutrient  medium  fails  to  grow  owing  to  the  antiseptic 
power  of  the  disinfectant  carried  over. 

METHODS  FOR  TESTING  DISINFECTANTS. 

(1).  .  Silk  threads.  This  method  was  introduced  by 
Koch  and  has  been  extensively  used.  Threads  of  silk,  linen, 
or  cotton  are  cut  up  into  lengths  of  about  1  cm.  They  are 
placed  in  a  sterilized  plugged  test-tube  and  sterilized  in 
the  dry-heat  oven.  A  cloudy  suspension  of  the  spores  or 
bacteria  to  be  tested  is  made  in  sterilized  water.  The 
pieces  of  sterilized  threads  are  immersed  in  this  suspen- 
sion for  some  minutes,  then  transferred  with  sterilized  for- 


MEMORANDA. 


18 


MEMORANDA. 


—  201  — 

ceps  to  a  sterilized  Petri  dish  and  allowed  to  dry.  They 
can  then  be  placed  in  a  test-tube  and  kept  for  future  use. 

To  ascertain  the  disinfecting  action  of  a  solution  a 
thread  impregnated  with  the  bacteria  to  be  tested  is  im- 
mersed in  it  fora  given  length  of  time,  as  for  instance  2 
minutes.  It  is  then  removed  with  sterilized  forceps  and 
gently  washed  in  sterilized  water  or  alcohol.  Finally  it  is 
transferred  to  a  tube  of  nutrient  bouillon  (10-15  c.  c.)  and 
then  set  aside  in  the  incubator  for  a  week  or  more.  Simi- 
lar tests  with  exposures  of  2,  5,  10,  3.0,  and  60  minutes 
should  be  made. 

The  objections  to  this  method  are  twofold  and  have 
already  been  incidentally  mentioned.  In  the  first  place 
the  bacteria  on  the  thread  may  not  be  evenly  exposed  to 
the  action  of  the  disinfectant  and  secondly  the  disinfec- 
tant itself  may  be  transferred  to  the  nutrient  medium. 
The  attempt  is  made  to  obviate  the  latter  objection  by 
washing  the  threads  and  while  this  may  be  successful 
in  some  cases,  in  others  it  fails.  Thus  mercuric  chloride 
is  apparently  held  fast  by  the  fibre  and  can  only  be  re- 
moved by  the  action  of  hydrogen  sulphide  (GEPPERT). 

(2).  Cover  glasses.  This  method  was  introduced  by 
Geppert  and  has  been  used  bySpirig  and  others.  Ordinary 
microscopic  cover-glasses  are  cut  in  two,  cleaned  and 
rendered  free  from  far,  and  finally  sterilized.  They  are 
then  immersed  in  the  bacterial  suspension,  or  in  bouillon 
cultures,  transferred  to  a  sterilized  wire  gauze,  under  a 
bell  jar,  and  allowed  to  dry.  To  test  a  disinfectant  a  dry 
cover-glasses  is  immersed  in  it  for  a  given  length  of  time 
as  in  the  case  of  the  silk  threads.  It  is  then  removed  with 
sterilized  forceps  and  washed  in  about  400  c.  c.  of  sterilized 
water  for  about  -|-|  hour.  Then  placed  in  sterilized  bouil- 
lon and  set  aside  in  the  incubator. 

The  advantages  of  this  method  are  (1)  that  a  thin  film 
of  evenly  spread  bacteria  is  employed,  and  (2)  that  the 
cover-glass  does  not  unite  with  the  disinfectant,  as  is  the 
case  with  the  silk  threads.  It  is  open  to  the  objection, 


—  202  — 

which  holds  true  also  for  the  silk  threads,  that  the  process 
of  desiccation  tends  to  lower  the  vitality  of  the  organism. 
Furthermore  it  may  be  urged  that  the  disinfectant  has  not 
free  access  to  all  sides  of  the  bacteria. 

(3.)  Bacterial  suspensions.  This  method  in  some  of 
its  modifications  is  the  one  which  is  commonly  employed 
and,  if  used  with  proper  precautions,  yields  perfectly  reli- 
able results.  The  first  essential  is  to  secure  a  suitable  sus- 
pension of  the  organism  to  be  tested.  For  this  purpose 
the  fresh  growth  on  the  surface  of  3  or  4  agar  tubes  is  care- 
fully removed  and  thoroughly  rubbed  up  in  about  10  c.  c.  of 
sterilized  distilled  water.  In  order  to  remove  the  coarse 
floccules  the  suspension  is  filtered  through  glass  wool,  and 
the  filtrate  immersed  in  a  water-bath  at  37.5°  C.  and  fre- 
quently agitated  till  a  microscopic  examination  shows  no 
longer  the  presence  of  groups  or  masses  of  bacteria.  In 
this  suspension  now  the  number  of  bacteria  present  can 
determined  as  already  stated. 

By  means  of  a  sterilized  pipette,  graduated  in  1-10  c.  c., 
an  exact  volume,  3  c.  c.,  is  transferred  into  each  of  several 
sterilized  test-tubes.  To  the  suspension  in  one  of  these 
tubes  an  equal  volume  of  the  disinfectant,  of  double  the 
strength  to  be  tested,  is  added.  At  intervals  of  2,  5, 10.  20, 
30,  60  minutes  etc.  transfers  are  made  to  sterilized  bouillon 
or  agar  tubes  and  these  are  then  set  aside  in  the  incubator 
for  at  least  one  week.  The  inoculations  should  be  made  in 
duplicate  and  2  or  3  loopfuls  used  for  each  tube. 

The  method  as  given  is  open  to  the  objection  that  an 
appreciable  amount  of  the  disinfectant  is  transferred  each 
time  to  the  culture  tubes  and  that  it  may  prevent  growth. 
This  is  specially  true  with  substances  which  possess 
marked  antiseptic  properties,  as  mercuric  chloride.  Where 
possible,  the  disinfectant  should  be  rendered  inert.  Thus, 
traces  of  mercuric  chloride  can  be  removed  by  precipita- 
tion with  hydrogen  sulphide.  With  other  substances  the 
error  is  not  so  marked  and  is  partly  counterbalanced  by 


MEMORANDA. 


MEMORANDA. 


—  203  — 
growing    the    tubes   in    the    incubator    for  many    days. 

(SCHAFFER.) 

LABORATORY  WORK. — The  student  should  test,  by  the 
methods  given,  the  disinfecting  action  of  mercuric  chloride 
(1-1000),  carbolic  acid  (5  per  cent),  hydrochloric  acid 
(0.4  per  cent.).  Anthrax  bacillus,  anthrax  spores,  staphy- 
lococcus  pyogenes  aureus,  cholera,  typhoid  fever  and 
dipththeria  bacilli  may  be  used,  suspended  in  distilled 
water,  bouillon  and  blood-serum. 

The  blood-serum  required  in  this  work  may  be  readily 
prepared  as  follows: — The  flowing  blood  from  an  ox  or 
calf  is  received  into  a  large  sterilized  Erlenmeyer  flask 
and  when  firmly  clotted  is  carried  to  the  laboratory  and 
placed  in  the  ice-chest.  After  24 — 48  hours  the  clear  yel- 
low serum  separates  out.  A  portion  of  this  may  be  placed 
in  a  beaker,  diluted  with  5—10  parts  of  distilled  water, 
then  filled  into  tubes  and  sterilized  as  in  the  case  of  or- 
dinary bouillon.  The  dilution  with  water  prevents  coagu- 
lation of  the  blood-serum.  Another  portion  of  the  blood- 
serum  may  be  filled  direct  in  tubes  which  are  then  placed 
in  an  inclined  position  in  an  air-bath  and  the  temperature 
slowly  raised  to  about  80°  C.  which  is  maintained  for 
about  one  hour.  The  serum  coagulates  in  the  inclined 
position.  On  the  following  day  the  tubes  should  be  steri- 
lized in  the  steam  sterilizer  for  ^— 1  hour. 

To  obtain  undiluted  fluid  blood-serum  the  blood  is  re- 
ceived directly  from  the  artery  or  vein  into  sterilized  jars 
or  flasks  which  are  protected  against  contamination  from 
the  air.  As  soon  as  the  serum  separates  it  is  transferred 
by  means  of  a  sterilized  pipette  to  the  sterilized  tubes. 
When  this  is  properly  done  no  organisms  are  introduced 
into  the  serum  and  hence  it  requires  no  subsequent  steri- 
lization. 

>i, 

OF  THE 

UNIVERSITY 
of 


MEMORANDA. 


MEMORANDA. 


MEMORANDA. 


—  205  — 


List  of  Apparatus  and  Aecessorie: 


1 
1 
1 

6 
1 
1 
2 

12 
12 
6 

H 

1 

100 
100 

3 
12 
12 

8 

200 
50 
100 

3 
100 

4 

1 


Flask,  2  litre. 
I     " 

"    y*   " 

"      50  c.c.  Erlenmeyer. 
Funnel,  15  cm.  diameter. 

6    " 

Moist  Chain  hers. 
Esma'ch  Dishes. 
Petri  Dishes. 

Staining  Di>hes,  with  covers. 
Watch  Glasses,  5  cm.  diarn. 
Test  Glass.  18cm.  high. 
Test  Tubes,  150x14  mm. 

125x12     " 
Tumblers. 
Glass  Plates. 
Glass  Benches. 
Glass  Rods,  18  cm. 
Cover  Glasses,  No.  1,  %  in.  diarn. 

Glass  Slides. 

Concave  Slides. 

Labels  for  Slides. 

Slide  Boxes. 

Slide  Disinfecting  Jar,  with  top, 

Six  10  cm. 
Disinfecting  Jar,  with  top,  15x20 

cm. 
Stain  bottles,  1  oz.  with  pipettes, 

in  stand. 

Glass  Pipettes,  1  c.c.  with  iron  box. 
Woltfhii^el  Colony  Counter. 
Novy  Bottle  for  Anaerobic  Tube 

Culture. 

Novy  Anaerobic  Plate  Apparatus. 
Cylinders,  graduated,  25, 100, 1000  c.c. 
Bottles,  2  oz. 
Waste  Dish. 
Wire  Gauze. 

Wire  Basket,  large  18x18x24  cm. 

"         "          small  10x12x18  cm. 

Iron  Sterilizing  Box,5xl4Uxl7V£ 

cm 

Bunsen  Burner,  with  tubing. 
Test  tube  Stand,  for  48  tubes. 
Support  Board,  large,  25x25  cm. 
"        small,  8x20  cm. 
Platinum  Wires,  No.  23,  5  cm, 
Pair  Pincers,  narrow  pointed,  10 

cm. 

Pair  Scissors,  14  cm. 
Scalpel. 
Potato  Knives. 
Colored  Wax  Pencil. 
Potato  brush. 
Iron  Water-bath,  with  tripod,  18 

cm.  diam. 

Wash-bottle,  siphon  or  bulb. 
Ice  apparatus  for  plates. 
Battery  Jars,  11x11  cm. 
Rat  Jars,  with  leaded  top. 


1  Crucible  Forceps. 

1  Chapman  Aspirator. 

1  Kipp's  hydrogen  generator. 

1  Koch  steam  Sterilizer,  with 

crown  ourner. 
1  Dry  Heat  Sterilizer,  with  crown 

burner. 

1  Incubator,  with  safety  lamp. 

2  Thermoregulatorss. 

1  Thermometer,  200°  C. 
1  "  00°  C. 

1  Microtome. 

1  Microscope,  with  3  objectives,  %, 
1-0,  and  1-12;  2  eyepieces;  Abbe 
condenser  and  iris  diaphragm. 
1  Roll  of  Cotton. 
12  Sheets  of  Filter  Paper. 

Rubber  caps. 

Fnchsine. 

Gentian  Violet. 

Methylene  Blue. 

Methyl  Violet. 

Bismarck  Brown. 

Kpsine. 

Picrocarrnine,  Weigert's. 

Anilin  Oil. 

Anilin  Hydrochloride. 

Iodine. 

Potassium  Iodide 

Mercuric  Chloride. 

Carbolic  Acid. 

Sulphuric  Acid. 

Nitric  Acid. 

Hydrochloric  Acid. 

Acetic  Acid. 

Pyrogallic  Acid. 

Tannic  Acid. 

Ferrous  Sulphate. 

Sodium  Carbonate. 

Sodium  Hydrate. 

Ammonium  Hydrate. 

Oil  of  Cloves. 

Oil  of  Cedar, 

Oil  of  Anise. 

Xylol 

Alcohol. 

Ether 

Chloroform. 

Paraffin,  40°,  46°,  52°,  56°  C. 

Collodium. 

Celloidin 

Sealing  Wax 

Tube  of  Canada  Balsam, 

Gelatin,  silver. 

Agar-Agar. 

Peptone^  sice.  Witte. 

Glucose. 

Glycerine. 

Litmus. 

Vaseline. 

Extract  of  Meat. 


ERRATA. 

Page  15. — Above  the  fifth  line  from  the  bottom  insert: 
Classification  according  to  oxygen   requirements— aerobic  and 
anaerobic. 

Gradations  in  requirements— facultative  and  obligative. 

Page  122.— On  the  23rd.  line  read  "Cultures  in" 

Page  123. — First  line,  read  "Culture"  instead  of  "Cultures." 


MEMORANDA. 


INDEX. 


Abbe  condenser.  12. 
Achorion.  126,  186. 
Aetinomyces,  126.  184. 
Aerobic  bacteria,  204. 
Aerogenic  bacteria,  17. 
Agar,  nutrient,  95. 

plates,  136. 
"      roll-tubes.  136. 
"      streak  cultures.  126. 
Air,  77. 

Alkaline  methylene  blue,  140. 
Amoeba  coli,  174. 

Anaerobic  apparatus  for  tubes,  124. 
"  *'  "    plates,  125. 

Anaerobic  bacteria,  116-122,  204. 

•*         culture  of,  123. 
Anilin  water,  106. 

"      fuchPine,  128. 
"      gentian  violet,  106. 
"      hydrochloride,  130,  194. 
Animal  inoculations,  101,  200. 
Animal  parasites,  100. 
Anthrax  bacillus,  114. 
"          sections,  191. 
"          work  with,  104. 
Arthrospore,  10. 
Asiatic  cholera,  148, 158. 
Aspergillus  flavescens,  88 
"  furnigatus,  90. 

"  niger,  88. 

Asporogenic  bacteria,  10. 
Attenuation,  100,  176,  199,  200. 
Bacillus,  5 

"        acidi  lactici,  66. 

"         anthracis,  114. 

"         butyrieus,  68. 

"        of  chicken  cholera,  176. 

•'         coli  communis,  158. 

"         cyanogenus,  70. 

"         diphtheria?,  140. 

"         fluorescens  putidus,  44. 

of  Friedlaehder,  144. 
•'         of  hog  cholera,  178. 
"        of  hog  erysipelas,  180. 

Indicus,  36. 
"         leprae,  134. 
mallei,  138. 
"         mega  teri  urn,  56. 
"        mesentericus  vulgatus,  54. 
"         murisepticus,  182. 
"         Neapolitanus,  156. 
"         oedernatis  rnaligni,  118. 


Bacterium,  5. 

coli  commune,  158. 
"  of  hog  cholera,  178. 

phosphorescens,  46. 
"  terrno,  60. 

"  Zopfli,  62. 

Black-leg,  116. 
Blue  milk,  70. 
Blue  pus,  168. 
Blood-serum,  203. 
Botkin  apparatus,  124. 
Bouillon,  95. 
Bread  flasks,  78. 
Buchner's  method,  124. 
Butyric  acid,  17,  68,  120. 
Calcium  hydrate  agar,  103. 
Capsule,  7,  142,  144,  172. 
Carbolic  fuchslne,  110. 
Caries,  dental,  18,  66. 
Cedar  oil,  12. 
Cell  wall,  7. 
Charbon,  114. 
Cheese  spirillum,  152. 
Chemistry  of  bacteria,  17. 
Chicken  cholera,  154, 176.  196. 

"        tuberculosis,  132. 
Chlorophyll,  7,  15. 
Cholera,  Asiatic,  148, 158. 

"         of  chicken,  154, 176. 

"        nostras,  150. 
Chromogenic  bacteria,  17. 
Classification,  5,  15,  17,  18,  94,  204. 

"  of  plants,  94. 

Clostridium,  10. 
Colony,  9,  21,97. 

"       examination  of  25. 
Comma  bacillus,  148. 
Concave  slide,  13. 
Condenser,  Abbe,  12. 
Cover-glasses,  12. 

"       "          preparation,  19,  106. 

"       "          for  disinfection,  201. 
Croupous  pneumonia,  142. 
Cutting  sections,  190. 
Deep  layer  cultures.  123, 125. 
Deneke's  bacillus,  152. 
Diaphragm,  Iris,  12. 
Diphtheria,  140. 

sections,  195. 
Diplococcus,  11. 

of  pneumonia,  142. 


Disinfection,  methods  of,  197,  200. 
No.  II,  120.Drum-stick  forms,  10. 

prodigiosus,  34.  Eberth's  bacillus,  160. 

pyocyaneus,  168.  Ebner's  solution,  194. 

ramosus,  58.  Emmerich's  bacillus,  156. 

of  rhinoscleroma,  146.  Endocarditis,  164,  166. 

rnber  of  Kiel,  38.  Endospore,  10. 

rubidus,  40.  Enzyme,  17. 

subtilis,  52.  Erysipelas,  162.  164. 

of  symptomatic  anthrax,  116.  Escherlch's  bacillus,  158. 

tetani,  122.  Esmarch  potato  culture,  30. 

"         tuberculosis,  132.  Esmarch  roll  culture,  30. 

"        typhi  abdominalis,  160.  Facultative,  15, 204. 

"        violaceus,  42.  Farcy,  138. 

Bacteria,  5,  94.  Favus,  126,  186. 


—  208 


Fermentation,  17. 
Kinkier-Prior's  bacillus,  150. 
Fhigellu,  7. 

'•         staining  of.  126. 
Flnoresfriuw  bacillus.  44. 
Frankel'sdiplococrus.  142,  l'.»5. 
FnetJleenUers  baeiliu.-,  144. 
Fungi,  5,  <M,  100. 
Gelatin,  niurient,  0. 

plates.  2!,  2). 
"         roll  cultures,  3'.). 

stich  cultures,  27. 
Generation,  10). 
Giant  whins,  7.  116-120. 
Glanders,  130,  138. 

"          sections,  19"). 
Glucose  agar.96. 

bouillon,  95. 
gelatin.  124. 
Glycerine  ayar,  9  i. 
Golden  pus  producing  coccus,  ICO. 
Gonococcus,  171). 
Gonorrhoea,  170. 
Gram's  method,  cover-glasses,  108. 

"  "          sections,  192. 

Granulose,  7. 
Green  pus,  108. 
Growth,  (Conditions  of,  15. 
Gruber's  tnl)es,  123. 
Hanging-drop  culture,  105. 

examination,  12. 
Hardening  tissue,  189. 
Hav  baciliu.s,  52. 
Herpes,  i?-6. 
liog  cholera,  173. 
Hog  erysipelas,  180. 
Hydrogen  cultures,  124,  125. 
Hydrogen  sulphide,  18,  122,  180. 
Hydrothionuria,  is. 
Impression  preparations,  GO. 
Indol  reaction,  148. 
Infection,  98. 

methods  of,  101. 
Infectious  diseases,  98 
Insolation,  10,  38. 
Intoxication,  98. 
Involution  forms  5. 
Iodine  solution,  107. 
Iris  diaphragm,  12 
Isolation  of  bacteria,  8.  21,  9!»,  104. 
'•  Klatsch  "  preparation's,  00. 
Lactic  acid,  17,  00. 
Leprosy,  134 

sections,  L94. 
Liborius  tubes,  124. 
Liebig's  meat' extract,  96. 
Liquefaction.  18. 
Litmus  media,  121. 
Lock-jaw,  122. 
Ldffler's  methyiene  blue,  140. 

mordant,  128. 
Lumpy-jaw,  120,  184. 
Lupus,  1 32 
Malignant  oedema,  1 18.         i 

sections,  193. 
Malignant  oedema,  No.  II,  12U 

•*.•-.  "  "       "    sect  ions,  194 

Malignant,  pustule,  111. 
Mai  lei n,  138. 
Meningitis,  142. 
Meningococcus.  142. 
Mercuric  chloride,  8,  189, 199. 
Microcorcus,  5. 

of  gonorrhoea,  170. 
pneumonias,  142. 
prodigiosus,  34. 
tetrasienus,  172. 
"  "  sections,  196. 


Microscope,  12. 

Micros  poron,  180. 

Mi  Ik,  75. 

Mixed  infection,  120,  144,  164. 

Moist  chamber.  8,  '24 

Monilia  Candida,  188. 

Mordant,  128. 

Morve,  138. 

Motion,  7 

Moulds,  78,  94. 

'•         culture  of.  7S 

"         examinalion  of,  78. 

Mouse  septicaemia,  182. 

Mucor  corymbifer.  84. 

rhizopodiformis.  8fi. 

Multiplication  of  bacteria,  10,  11. 

Tsitrification.  18. 

Non  ])athogenic  bacteria,  1^. 

Non-toxicogenic  bac;cria,  IS. 

Nucleus,  7. 

Qiiligative,  15,204 

<  >bjectives,  12. 

(Edema,  malignant.  118,  120. 

Oidium  albicans,    88. 
lactis,  72. 

Orange  sarcine.  48. 

Osteomyelitis,  HJ ,. 

Paraffin  sections,  190 

Parasitic  bacteria,  15. 

Pathogenic  bacteria,  18.  97. 

Penicillium  glaucum,  82. 

Pericarditis,  142. 

Peritonitis,  142. 

Petri  dish  cultures,  29. 

Pfeiffer's  method,  192. 

Phagocytes,  li.l. 

l>hosphore^cence,  IS,  46. 

Photobacterium,  46 

Phoiogenic  bacteria,  17. 

Pigment,  18,04. 

Pityriasis,  180. 

Plasmodium,  174. 

Plants.  94. 

Plates,  sterilization  of,  22,  29. 

Plates,  culture  on,  21,  29,  130. 

Pleuritis,  142. 

Pneumococcus,  144. 

Pneumonia,  142,  144,  195. 

Poisoned  arrows,  110,  1 18,  122. 

Post-mortem  examination,  103. 

Potato  bacillus,  54. 

cultures.  7,  30. 
"       Esmarch  cultures,  30. 
"       tube  culture,  31. 

Precautions. !»,  102,  101. 

Proteids,  17. 

Proteus  vulgaris,  00. 

Ptomaines,  17. 

Puerperal  fever,  161. 

Pure  culture,  9,  21,26,  97. 

Putrefaction,  18. 

Pya>miu,  104 

sections.  196. 

Pyrogallate  met  hod,  124,  125. 

Quarter-evil,  1 10. 

Kag-picker'-s  disease.  114,  118. 

Kay-fungus,  184. 

Recurrent  lever,  171 

Red  bacillus  of  Kiel,  38. 
".    \vater,  40. 

Ked  yeast,  92. 

Relapsing  lever,  174. 

Reproduction,  of  bacteria,  10. 

Khinoscleroma,  146. 

Roll  culture,  agar,  136. 
gelatin,  30. 

Root  bacillus,  53. 

Rouget,  J80. 


—  209  — 


Rules  of  Koch,  98. 
Saccharomyces,  79,  188. 
Sjiliva.  21,  30,  142,  144,  172. 
Saprogenic  bacteria,  17. 
Saprophytic  bacteria,  15. 
Sarcine,  11. 

"         orange,  48. 
"         yellow,  50. 
Sections,  "89. 

"         Gram's  stain,  192. 
"         simple  stain,  191. 
"          tubercle,  194. 
Septicemie,  118. 
Silk  threads,  198,200. 
Soil,  7(i. 
Spirillum,  5. 

"  Oberrneieri,  174. 

rubrurn,  64. 
"  tyrogenum,  152. 

Spirocheete,  174. 
Splenic  fever,  114. 
Spontaneous  generation,  15. 
Spores,  10,  109,  197. 
Spores,  double  stain,  109. 
Sporogenic  granules,  10,  105. 
Sporozoa,  100. 
Sputum,  130,  142,  144,172. 
Sputum  septica3mia,  142. 
Siaining  coverglasses,  simple,  19,  106. 

Gram's,  106. 
"  tubercle,  130. 

Staining  sections,  191. 
Stains,  19. 
Staphylococcus.  11. 

"  pyog.  albus,  166. 

"      aureus,  166, 196. 
"      citreus.  166. 
Sterilization  of  media,  6,  8,  78,  96,  203. 

"  plates,  22,  29. 
"  "  tubes,  6. 

Stich  cultures,  27. 
Streak  cultures,  32, 126. 
Streak  preparations,  106. 
Streptococcus,  11. 

erysipelatis,  162. 
pyogenes,  164,  196. 


Summer  diarrhoea,  18. 

Suppuration,  162-172. 

Suspensions  for  disinfection,  202. 

Swine  plague,  178. 

Symptomatic  anthrax,  116, 194. 

Temperature.  15,  198. 

Testing  disinfectants,  200. 

Tetanus,  122. 

Tetrads.  H. 

Threads,  11. 

Threads  for  disinfection,  198. 

Thrush,  1 88 

Toxicogenic  bacteria,  18. 

Tricophyton,  186. 

Tubercle  bacillus,  126,  130,132. 

sections,  194.     ; 
sputum,  130. 
Tuberculin,  1&2. 
Tuberculosis  of  chicken,  132. 
Typhoid  fever,  160,  195. 
TTrine,  fermentation  of,  18. 
Vficuum  cultures,  123,  125. 
Vibrio,  5. 

of  Asiatic  cholera,  148. 
of  Deneke,  152. 
of  Finkler-Prior,  150. 
Metchnikovi,  154,  176. 
proteus,  150. 
Vibrion  butyrique,  68. 
Vibrion  septique,  118. 
Violet  bacillus  of  water,  42. 
Water,  73. 
Whips,  7. 

Wool-sorter's  disease,  114. 
Wurzel  bacillus,  58. 
Yeast,  5,  94. 

"       baker's,  79. 

"       black,  79. 

"      red,  79,  92. 

"      white,  79. 
Yellow  sarcine,  50. 
Ziehl-Neelsen  method,  130. 
Ziehl's  solution,  110. 
Zoogloea,  7. 
Zymogenic  bacteria,  17. 


MEMORANDA. 


BOOK  T      ,„.   ^ 


132699 


JUBRARY 
G 


i 


THE  I 


r  "  *  fI70IJ 


TBRARY 


